Penicillin-g acylases

ABSTRACT

The present invention provides engineered penicillin G acylase (PGA) enzymes, polynucleotides encoding the enzymes, compositions comprising the enzymes, and methods of using the engineered PGA enzymes.

The present application is a divisional of U.S. patent application Ser. No. 15/861,849, filed Jan. 4, 2018, which claims priority to US Pat. Appln. Ser. Nos. 62/442,810 and 62/472,055, filed on Jan. 5, 2017 and Mar. 16, 2017, respectively, all of which are hereby incorporated by reference in their entireties for all purposes.

FIELD OF THE INVENTION

The present invention provides engineered penicillin G acylase (PGA) enzymes, polynucleotides encoding the enzymes, compositions comprising the enzymes, and methods of using the engineered PGA enzymes.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The official copy of the Sequence Listing is submitted concurrently with the specification as an ASCII formatted text file via EFS-Web, with a file name of “CX2-161USP2_ST25.txt”, a creation date of Feb. 15, 2017, and a size of 940 kilobytes. The Sequence Listing filed via EFS-Web is part of the specification and is incorporated in its entirety by reference herein.

BACKGROUND OF THE INVENTION

Penicillin G acylase (PGA) (penicillin amidase, EC 3.5.1.11) catalyzes the cleavage of the amide bond of the penicillin G (benzylpenicillin) side chain. The enzyme is used commercially in the manufacture of 6-amino-penicillanic acid (6-APA) and phenyl-acetic acid (PAA). 6-APA is a key compound in the industrial production of semi-synthetic β-lactam antibiotics such as amoxicillin, ampicillin and cephalexin. The naturally occurring PGA enzyme shows instability in commercial processes, requiring immobilization on solid substrates for commercial applications. PGA has been covalently bonded to various supports and PGA immobilized systems have been reported as useful tools for the synthesis of pure optical isomers. Attachment to solid surfaces, however, leads to compromised enzyme properties, such as reduced activity and/or selectivity, and limitations to solute access. Moreover, although attachment to solid substrates allows capture of enzymes and reuse in additional processing cycles, the stability of the enzyme is such that such applications may be limited. The enzymatic catalysis by PGA of penicillin G to 6-APA is regiospecific (it does not cleave the lactam amide bond) and stereospecific. The production of 6-APA constitutes perhaps the largest utilization of enzymatic catalysis in the production of pharmaceuticals. The enzymatic activity of PGA, associated with the phenacetyl moiety, allows the stereospecific hydrolysis of a rich variety of phenacetyl derivatives of primary amines as well as alcohols.

SUMMARY OF THE INVENTION

The present invention provides engineered penicillin G acylase (PGA) enzymes, polynucleotides encoding the enzymes, compositions comprising the enzymes, and methods of using the engineered PGA enzymes. The present invention provides engineered penicillin G acylases capable of acylating insulin, wherein the polypeptide sequence of said penicillin G acylase is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:2, 4, 12, 24, 40, 56, 70, 82, 100, 108, 110, 116, 136, 142, 154 and 160. In some embodiments, penicillin G acylase comprises SEQ ID NO: 4, 12, 24, 40, 56, 70, 82, 108, 110, 116, 136, 142, 154, or 160. In some further embodiments, the engineered penicillin G acylase comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to at least one sequence set forth in Table 5.1, 5.1, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 7.1, 8.1, 9.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, and/or Table 20.1. In some additional embodiments, the engineered penicillin G acylase comprises a sequence comprises a sequence set forth in Table 5.1, 5.1, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 7.1, 8.1, 9.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, and/or Table 20.1. In still some further embodiments, the engineered penicillin G acylase comprises a histidine tag. In some embodiments, the histidine tag is present at the C-terminus of the engineered penicillin G acylase. In some additional embodiments, the engineered penicillin G acylase comprises a polypeptide sequence selected from SEQ ID NO:110 and SEQ ID NO:142.

The present invention also provides engineered polynucleotide sequences encoding the engineered penicillin G acylases provided herein. In some embodiments, the engineered polynucleotide sequence comprises a polynucleotide sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NOS: 1, 11, 23, 39, 55, 69, 81, 99, 107, 109, 115, 135, 141, 153, and/or 159.

The present invention also provides vectors comprising the engineered polynucleotide sequences provided herein. In some embodiments, the vectors comprise at least one engineered polynucleotide sequence comprising polynucleotide sequence(s) at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NOS: 2, 11, 23, 39, 55, 69, 81, 99, 107, 109, 115, 135, 141, 153, and/or 159. In some further embodiments, the vectors comprise at least one control sequence.

The present invention also provides host cells comprising at least one vector provided herein. In some embodiments, the vectors within the host cells comprise at least one engineered polynucleotide sequence comprising polynucleotide sequence(s) at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NOS: 1, 11, 23, 39, 55, 69, 81, 99, 107, 109, 115, 135, 141, 153, and/or 159. In some further embodiments, the vectors comprise at least one control sequence.

The present invention also provides methods for producing acylated insulin, comprising providing: at least one engineered penicillin G acylase of any of claims 1-7 and insulin; and exposing the engineered penicillin G acylase and insulin under conditions such that the engineered penicillin G acylase acrylates the insulin, thereby producing acylated insulin. In some embodiments, the acylation is conducted in the presence of methyl phenylacetate. In some further embodiments, acylation occurs at any of positions A1, B1, and/or B29 of said insulin. In some additional embodiments, acylation occurs at position A1 of said insulin, while in some alternative embodiments, acylation occurs at position B1 of said insulin, and in still other embodiments, acylation occurs at position B29 of said insulin. In some embodiments, acylation occurs at positions A1, B1, and/or B29 of said insulin. In some further embodiments, acylation occurs at positions A1, B1, and B29 of the insulin. In still some additional embodiments of the methods, the engineered penicillin G acylase produces more than 90% more acylated insulin as compared to the production of acylated insulin by the polypeptide of SEQ ID NO:2, 4, 12, 24, 40, 56, 70, 82, 100, 108, 110, 116, 136, 142, 154 and/or 160.

The present invention also provides acylated insulin compositions produced using at least one engineered penicillin G acylase provided herein. In some additional embodiments, the present invention provides compositions comprising acylated insulin produced using at least one method provided herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a chromatogram of the analytical method described in Table 21.5 used to quantify insulin and the elution order of the acylated products.

FIG. 2 provides the results of the experiments described in Example 10.

FIG. 3 provides the results of the experiments described in Example 16.

DESCRIPTION OF THE INVENTION

The present invention provides engineered penicillin G acylases (PGA) that are capable of cleaving penicillin to phenylacetic acid and 6-aminopenicillanic acid (6-APA), which is a key intermediate in the synthesis of a large variety of β-lactam antibiotics. In particular, the present invention provides engineered PGAs that are capable of producing phenyl acetate mono-protected or di-protected insulin by adding the protecting group to the A1, B1 or B29 positions of free insulin or removing protecting groups from A1/B1/B29 tri-protected insulin or removing the A1/B1/B29 tri-phenyl acetate protecting groups to release free insulin.

Generally, naturally occurring PGAs are heterodimeric enzymes composed of an alpha subunit and a beta-subunit. Wild-type PGA is naturally synthesized as a pre-pro-PGA polypeptide, containing an N-terminal signal peptide that mediates translocation to the periplasm and a linker region connecting the C-terminus of the alpha subunit to the N-terminus of the beta subunit. Proteolytic processing leads to the mature heterodimeric enzyme. The intermolecular linker region can also function in promoting proper folding of the enzyme. The PGAs provided herein are based on the PGA from Kluyvera citrophila in which various modifications have been introduced to generate improved enzymatic properties as described in detail below.

For the descriptions provided herein, the use of the singular includes the plural (and vice versa) unless specifically stated otherwise. For instance, the singular forms “a”, “an” and “the” include plural referents unless the context clearly indicates otherwise. Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.”

Both the foregoing general description, including the drawings, and the following detailed description are exemplary and explanatory only and are not restrictive of this disclosure. Moreover, the section headings used herein are for organizational purposes only and not to be construed as limiting the subject matter described.

Definitions

As used herein, the following terms are intended to have the following meanings.

In reference to the present disclosure, the technical and scientific terms used in the descriptions herein will have the meanings commonly understood by one of ordinary skill in the art, unless specifically defined otherwise. Accordingly, the following terms are intended to have the following meanings. All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference. Unless otherwise indicated, the practice of the present invention involves conventional techniques commonly used in molecular biology, fermentation, microbiology, and related fields, which are known to those of skill in the art. Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. Indeed, it is intended that the present invention not be limited to the particular methodology, protocols, and reagents described herein, as these may vary, depending upon the context in which they are used. The headings provided herein are not limitations of the various aspects or embodiments of the present invention.

Nonetheless, in order to facilitate understanding of the present invention, a number of terms are defined below. Numeric ranges are inclusive of the numbers defining the range. Thus, every numerical range disclosed herein is intended to encompass every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. It is also intended that every maximum (or minimum) numerical limitation disclosed herein includes every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were expressly written herein.

As used herein, the term “comprising” and its cognates are used in their inclusive sense (i.e., equivalent to the term “including” and its corresponding cognates).

As used herein and in the appended claims, the singular “a”, “an” and “the” include the plural reference unless the context clearly dictates otherwise. Thus, for example, reference to a “host cell” includes a plurality of such host cells.

Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation and amino acid sequences are written left to right in amino to carboxy orientation, respectively.

The headings provided herein are not limitations of the various aspects or embodiments of the invention that can be had by reference to the specification as a whole. Accordingly, the terms defined below are more fully defined by reference to the specification as a whole.

As used herein, the terms “protein,” “polypeptide,” and “peptide” are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristilation, ubiquitination, etc.). Included within this definition are D- and L-amino acids, and mixtures of D- and L-amino acids.

As used herein, “polynucleotide” and “nucleic acid’ refer to two or more nucleosides that are covalently linked together. The polynucleotide may be wholly comprised ribonucleosides (i.e., an RNA), wholly comprised of 2′ deoxyribonucleotides (i.e., a DNA) or mixtures of ribo- and 2′ deoxyribonucleosides. While the nucleosides will typically be linked together via standard phosphodiester linkages, the polynucleotides may include one or more non-standard linkages. The polynucleotide may be single-stranded or double-stranded, or may include both single-stranded regions and double-stranded regions. Moreover, while a polynucleotide will typically be composed of the naturally occurring encoding nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), it may include one or more modified and/or synthetic nucleobases (e.g., inosine, xanthine, hypoxanthine, etc.). Preferably, such modified or synthetic nucleobases will be encoding nucleobases.

As used herein, “hybridization stringency” relates to hybridization conditions, such as washing conditions, in the hybridization of nucleic acids. Generally, hybridization reactions are performed under conditions of lower stringency, followed by washes of varying but higher stringency. The term “moderately stringent hybridization” refers to conditions that permit target-DNA to bind a complementary nucleic acid that has about 60% identity, preferably about 75% identity, about 85% identity to the target DNA; with greater than about 90% identity to target-polynucleotide. Exemplary moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5×Denhart's solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE, 0.2% SDS, at 42° C. “High stringency hybridization” refers generally to conditions that are about 10° C. or less from the thermal melting temperature T, as determined under the solution condition for a defined polynucleotide sequence. In some embodiments, a high stringency condition refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C. (i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will not be stable under high stringency conditions, as contemplated herein). High stringency conditions can be provided, for example, by hybridization in conditions equivalent to 50% formamide, 5×Denhart's solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE, and 0.1% SDS at 65° C. Another high stringency condition is hybridizing in conditions equivalent to hybridizing in 5×SSC containing 0.1% (w:v) SDS at 65° C. and washing in 0.1×SSC containing 0.1% SDS at 65° C. Other high stringency hybridization conditions, as well as moderately stringent conditions, are known to those of skill in the art.

As used herein, “coding sequence” refers to that portion of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein.

As used herein, “codon optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest. In some embodiments, the polynucleotides encoding the PGA enzymes may be codon optimized for optimal production from the host organism selected for expression. Although the genetic code is degenerate in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, the polynucleotides encoding the PGAs enzymes may be codon optimized for optimal production from the host organism selected for expression.

As used herein, “preferred, optimal, high codon usage bias codons” refers interchangeably to codons that are used at higher frequency in the protein coding regions than other codons that code for the same amino acid. The preferred codons may be determined in relation to codon usage in a single gene, a set of genes of common function or origin, highly expressed genes, the codon frequency in the aggregate protein coding regions of the whole organism, codon frequency in the aggregate protein coding regions of related organisms, or combinations thereof. Codons whose frequency increases with the level of gene expression are typically optimal codons for expression. A variety of methods are known for determining the codon frequency (e.g., codon usage, relative synonymous codon usage) and codon preference in specific organisms, including multivariate analysis, for example, using cluster analysis or correspondence analysis, and the effective number of codons used in a gene (See e.g., GCG CodonPreference, Genetics Computer Group Wisconsin Package; Codon W, John Peden, University of Nottingham; McInerney, Bioinform., 14:372-73 [1998]; Stenico et al., Nucleic Acids Res., 222:437-46 [1994]; and Wright, Gene 87:23-29 [1990]). Codon usage tables are available for a growing list of organisms (See e.g., Wada et al., Nucleic Acids Res., 20:2111-2118 [1992]; Nakamura et al., Nucl. Acids Res., 28:292 [2000]; Duret, et al., supra; Henaut and Danchin, “Escherichia coli and Salmonella,” Neidhardt, et al. (eds.), ASM Press, Washington D.C., [1996], p. 2047-2066. The data source for obtaining codon usage may rely on any available nucleotide sequence capable of coding for a protein. These data sets include nucleic acid sequences actually known to encode expressed proteins (e.g., complete protein coding sequences-CDS), expressed sequence tags (ESTS), or predicted coding regions of genomic sequences (See e.g., Uberbacher, Meth. Enzymol., 266:259-281 [1996]; Tiwari et al., Comput. Appl. Biosci., 13:263-270 [1997]).

As used herein, “control sequence” is defined herein to include all components, which are necessary or advantageous for the expression of a polynucleotide and/or polypeptide of the present invention. Each control sequence may be native or foreign to the polynucleotide of interest. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator.

As used herein, “operably linked” is defined herein as a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) at a position relative to a polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide and/or polypeptide of interest.

As used herein, “promoter sequence” refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The control sequence may comprise an appropriate promoter sequence. The promoter sequence contains transcriptional control sequences, which mediate the expression of a polynucleotide of interest. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

As used herein, “naturally occurring” or “wild-type” refers to the form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation.

As used herein, “non-naturally occurring,” “engineered,” and “recombinant” when used in the present disclosure with reference to (e.g., a cell, nucleic acid, or polypeptide), refers to a material, or a material corresponding to the natural or native form of the material, that has been modified in a manner that would not otherwise exist in nature. In some embodiments the material is identical to naturally occurring material, but is produced or derived from synthetic materials and/or by manipulation using recombinant techniques. Non-limiting examples include, among others, recombinant cells expressing genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise expressed at a different level.

As used herein, “percentage of sequence identity,” “percent identity,” and “percent identical” refer to comparisons between polynucleotide sequences or polypeptide sequences, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Determination of optimal alignment and percent sequence identity is performed using the BLAST and BLAST 2.0 algorithms (See e.g., Altschul et al., J. Mol. Biol. 215: 403-410 [1990]; and Altschul et al., Nucl. Acids Res. 3389-3402 [1977]). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website.

Briefly, the BLAST analyses involve first identifying high scoring sequence pairs (HSPs) by identifying short words of length Win the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (See e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 [1989]).

Numerous other algorithms are available and known in the art that function similarly to BLAST in providing percent identity for two sequences. Optimal alignment of sequences for comparison can be conducted using any suitable method known in the art (e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 [1981]; by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 [1970]; by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988]; and/or by computerized implementations of these algorithms [GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package]), or by visual inspection, using methods commonly known in the art. Additionally, determination of sequence alignment and percent sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison Wis.), using the default parameters provided.

As used herein, “substantial identity” refers to a polynucleotide or polypeptide sequence that has at least 80 percent sequence identity, at least 85 percent identity and 89 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 residue positions, frequently over a window of at least 30-50 residues, wherein the percentage of sequence identity is calculated by comparing the reference sequence to a sequence that includes deletions or additions which total 20 percent or less of the reference sequence over the window of comparison. In specific embodiments applied to polypeptides, the term “substantial identity” means that two polypeptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 89 percent sequence identity, at least 95 percent sequence identity or more (e.g., 99 percent sequence identity). In some preferred embodiments, residue positions that are not identical differ by conservative amino acid substitutions.

As used herein, “reference sequence” refers to a defined sequence to which another sequence is compared. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotide or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, or the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides over a comparison window to identify and compare local regions of sequence similarity. The term “reference sequence” is not intended to be limited to wild-type sequences, and can include engineered or altered sequences. For example, in some embodiments, a “reference sequence” can be a previously engineered or altered amino acid sequence.

As used herein, “comparison window” refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The comparison window can be longer than 20 contiguous residues, and includes, optionally 30, 40, 50, 100, or longer windows.

As used herein, “corresponding to,” “reference to,” and “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of an engineered PGA, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned. As used herein, a reference to a residue position, such as “Xn” as further described below, is to be construed as referring to “a residue corresponding to”, unless specifically denoted otherwise. Thus, for example, “X94” refers to any amino acid at position 94 in a polypeptide sequence.

As used herein, “improved enzyme property” refers to a PGA that exhibits an improvement in any enzyme property as compared to a reference PGA. For the engineered PGA polypeptides described herein, the comparison is generally made to the wild-type PGA enzyme, although in some embodiments, the reference PGA can be another improved engineered PGA. Enzyme properties for which improvement is desirable include, but are not limited to, enzymatic activity (which can be expressed in terms of percent conversion of the substrate at a specified reaction time using a specified amount of PGA), chemoselectivity, thermal stability, solvent stability, pH activity profile, cofactor requirements, refractoriness to inhibitors (e.g., product inhibition), stereospecificity, and stereoselectivity (including enantioselectivity).

As used herein, “increased enzymatic activity” refers to an improved property of the engineered PGA polypeptides, which can be represented by an increase in specific activity (e.g., product produced/time/weight protein) or an increase in percent conversion of the substrate to the product (e.g., percent conversion of starting amount of substrate to product in a specified time period using a specified amount of PGA) as compared to the reference PGA enzyme. Exemplary methods to determine enzyme activity are provided in the Examples. Any property relating to enzyme activity may be affected, including the classical enzyme properties of K_(m), V_(max) or k_(cat), changes of which can lead to increased enzymatic activity. Improvements in enzyme activity can be from about 1.5 times the enzymatic activity of the corresponding wild-type PGA enzyme, to as much as 2 times. 5 times, 10 times, 20 times, 25 times, 50 times, 75 times, 100 times, or more enzymatic activity than the naturally occurring PGA or another engineered PGA from which the PGA polypeptides were derived. In specific embodiments, the engineered PGA enzyme exhibits improved enzymatic activity in the range of 1.5 to 50 times, 1.5 to 100 times greater than that of the parent PGA enzyme. It is understood by the skilled artisan that the activity of any enzyme is diffusion limited such that the catalytic turnover rate cannot exceed the diffusion rate of the substrate, including any required cofactors. The theoretical maximum of the diffusion limit, or k_(cat)/K_(m), is generally about 10⁸ to 10⁹ (M⁻¹ s⁻¹). Hence, any improvements in the enzyme activity of the PGA will have an upper limit related to the diffusion rate of the substrates acted on by the PGA enzyme. PGA activity can be measured by any one of standard assays used for measuring the release of phenylacetic acid upon cleavage of penicillin G, such as by titration (See e.g., Simons and Gibson, Biotechnol. Tech., 13:365-367 [1999]). In some embodiments, the PGA activity can be measured by using 6-nitrophenylacetamido benzoic acid (NIPAB), which cleavage product 5-amino-2-nitro-benzoic acid is detectable spectrophotometrically (λmax=405 nm). Comparisons of enzyme activities are made using a defined preparation of enzyme, a defined assay under a set condition, and one or more defined substrates, as further described in detail herein. Generally, when lysates are compared, the numbers of cells and the amount of protein assayed are determined as well as use of identical expression systems and identical host cells to minimize variations in amount of enzyme produced by the host cells and present in the lysates.

As used herein, “increased enzymatic activity” and “increased activity” refer to an improved property of an engineered enzyme, which can be represented by an increase in specific activity (e.g., product produced/time/weight protein) or an increase in percent conversion of the substrate to the product (e.g., percent conversion of starting amount of substrate to product in a specified time period using a specified amount of PGA) as compared to a reference enzyme as described herein. Any property relating to enzyme activity may be affected, including the classical enzyme properties of K_(m), V_(max) or k_(cat), changes of which can lead to increased enzymatic activity. In some embodiments, the PGA enzymes provided herein frees insulin by removing tri-phenyl acetate protecting groups from specific residues of insulin. Comparisons of enzyme activities are made using a defined preparation of enzyme, a defined assay under a set condition, and one or more defined substrates, as further described in detail herein. Generally, when enzymes in cell lysates are compared, the numbers of cells and the amount of protein assayed are determined as well as use of identical expression systems and identical host cells to minimize variations in amount of enzyme produced by the host cells and present in the lysates.

As used herein, “conversion” refers to the enzymatic transformation of a substrate to the corresponding product.

As used herein “percent conversion” refers to the percent of the substrate that is converted to the product within a period of time under specified conditions. Thus, for example, the “enzymatic activity” or “activity” of a PGA polypeptide can be expressed as “percent conversion” of the substrate to the product.

As used herein, “chemoselectivity” refers to the preferential formation in a chemical or enzymatic reaction of one product over another.

As used herein, “thermostable” and “thermal stable” are used interchangeably to refer to a polypeptide that is resistant to inactivation when exposed to a set of temperature conditions (e.g., 40-80° C.) for a period of time (e.g., 0.5-24 hrs) compared to the untreated enzyme, thus retaining a certain level of residual activity (e.g., more than 60% to 80%) after exposure to elevated temperatures.

As used herein, “solvent stable” refers to the ability of a polypeptide to maintain similar activity (e.g., more than e.g., 60% to 80%) after exposure to varying concentrations (e.g., 5-99%) of solvent (e.g., isopropyl alcohol, tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butylacetate, methyl tert-butylether, etc.) for a period of time (e.g., 0.5-24 hrs) compared to the untreated enzyme.

As used herein, “pH stable” refers to a PGA polypeptide that maintains similar activity (e.g., more than 60% to 80%) after exposure to high or low pH (e.g., 4.5-6 or 8 to 12) for a period of time (e.g., 0.5-24 hrs) compared to the untreated enzyme.

As used herein, “thermo- and solvent stable” refers to a PGA polypeptide that is both thermostable and solvent stable.

As used herein, “hydrophilic amino acid or residue” refers to an amino acid or residue having a side chain exhibiting a hydrophobicity of less than zero according to the normalized consensus hydrophobicity scale of Eisenberg et al., (Eisenberg et al., J. Mol. Biol., 179:125-142 [1984]). Genetically encoded hydrophilic amino acids include L-Thr (T), L-Ser (S), L-His (H), L-Glu (E), L-Asn (N), L-Gln (Q), L-Asp (D), L-Lys (K) and L-Arg (R).

As used herein, “acidic amino acid or residue” refers to a hydrophilic amino acid or residue having a side chain exhibiting a pK value of less than about 6 when the amino acid is included in a peptide or polypeptide. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Genetically encoded acidic amino acids include L-Glu (E) and L-Asp (D).

As used herein, “basic amino acid or residue” refers to a hydrophilic amino acid or residue having a side chain exhibiting a pK value of greater than about 6 when the amino acid is included in a peptide or polypeptide. Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ion. Genetically encoded basic amino acids include L-Arg (R) and L-Lys (K).

As used herein, “polar amino acid or residue” refers to a hydrophilic amino acid or residue having a side chain that is uncharged at physiological pH, but which has at least one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Genetically encoded polar amino acids include L-Asn (N), L-Gln (Q), L-Ser (S) and L-Thr (T).

As used herein, “hydrophobic amino acid or residue” refers to an amino acid or residue having a side chain exhibiting a hydrophobicity of greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg et al., (Eisenberg et al., J. Mol. Biol., 179:125-142 [1984]). Genetically encoded hydrophobic amino acids include L-Pro (P), L-Ile (I), L-Phe (F), L-Val (V), L-Leu (L), L-Trp (W), L-Met (M), L-Ala (A) and L-Tyr (Y).

As used herein, “aromatic amino acid or residue” refers to a hydrophilic or hydrophobic amino acid or residue having a side chain that includes at least one aromatic or heteroaromatic ring. Genetically encoded aromatic amino acids include L-Phe (F), L-Tyr (Y) and L-Trp (W). Although owing to the pKa of its heteroaromatic nitrogen atom L-His (H) it is sometimes classified as a basic residue, or as an aromatic residue as its side chain includes a heteroaromatic ring, herein histidine is classified as a hydrophilic residue or as a “constrained residue” (see below).

As used herein, “constrained amino acid or residue” refers to an amino acid or residue that has a constrained geometry. Herein, constrained residues include L-Pro (P) and L-His (H). Histidine has a constrained geometry because it has a relatively small imidazole ring. Proline has a constrained geometry because it also has a five membered ring.

As used herein, “non-polar amino acid or residue” refers to a hydrophobic amino acid or residue having a side chain that is uncharged at physiological pH and which has bonds in which the pair of electrons shared in common by two atoms is generally held equally by each of the two atoms (i.e., the side chain is not polar). Genetically encoded non-polar amino acids include L-Gly (G), L-Leu (L), L-Val (V), L-Ile (I), L-Met (M) and L-Ala (A).

As used herein, “aliphatic amino acid or residue” refers to a hydrophobic amino acid or residue having an aliphatic hydrocarbon side chain. Genetically encoded aliphatic amino acids include L-Ala (A), L-Val (V), L-Leu (L) and L-Ile (I).

It is noted that cysteine (or “L-Cys” or “[C]”) is unusual in that it can form disulfide bridges with other L-Cys (C) amino acids or other sulfanyl- or sulfhydryl-containing amino acids. The “cysteine-like residues” include cysteine and other amino acids that contain sulfhydryl moieties that are available for formation of disulfide bridges. The ability of L-Cys (C) (and other amino acids with —SH containing side chains) to exist in a peptide in either the reduced free —SH or oxidized disulfide-bridged form affects whether L-Cys (C) contributes net hydrophobic or hydrophilic character to a peptide. While L-Cys (C) exhibits a hydrophobicity of 0.29 according to the normalized consensus scale of Eisenberg (Eisenberg et al., 1984, supra), it is to be understood that for purposes of the present disclosure, L-Cys (C) is categorized into its own unique group.

As used herein, “small amino acid or residue” refers to an amino acid or residue having a side chain that is composed of a total three or fewer carbon and/or heteroatoms (excluding the α-carbon and hydrogens). The small amino acids or residues may be further categorized as aliphatic, non-polar, polar or acidic small amino acids or residues, in accordance with the above definitions. Genetically-encoded small amino acids include L-Ala (A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T) and L-Asp (D).

As used herein, “hydroxyl-containing amino acid or residue” refers to an amino acid containing a hydroxyl (—OH) moiety. Genetically-encoded hydroxyl-containing amino acids include L-Ser (S) L-Thr (T) and L-Tyr (Y).

As used herein, “amino acid difference” and “residue difference” refer to a difference in the amino acid residue at a position of a polypeptide sequence relative to the amino acid residue at a corresponding position in a reference sequence. The positions of amino acid differences generally are referred to herein as “Xn,” where n refers to the corresponding position in the reference sequence upon which the residue difference is based. For example, a “residue difference at position X40 as compared to SEQ ID NO:2” refers to a difference of the amino acid residue at the polypeptide position corresponding to position 40 of SEQ ID NO:2. Thus, if the reference polypeptide of SEQ ID NO:2 has a histidine at position 40, then a “residue difference at position X40 as compared to SEQ ID NO:2” refers to an amino acid substitution of any residue other than histidine at the position of the polypeptide corresponding to position 40 of SEQ ID NO:2. In most instances herein, the specific amino acid residue difference at a position is indicated as “XnY” where “Xn” specified the corresponding position as described above, and “Y” is the single letter identifier of the amino acid found in the engineered polypeptide (i.e., the different residue than in the reference polypeptide). In some instances, the present disclosure also provides specific amino acid differences denoted by the conventional notation “AnB”, where A is the single letter identifier of the residue in the reference sequence, “n” is the number of the residue position in the reference sequence, and B is the single letter identifier of the residue substitution in the sequence of the engineered polypeptide. In some instances, a polypeptide of the present disclosure can include one or more amino acid residue differences relative to a reference sequence, which is indicated by a list of the specified positions where residue differences are present relative to the reference sequence. In some embodiments, where more than one amino acid can be used in a specific residue position of a polypeptide, the various amino acid residues that can be used are separated by a “/” (e.g., X192A/G). The present disclosure includes engineered polypeptide sequences comprising one or more amino acid differences that include either/or both conservative and non-conservative amino acid substitutions. The amino acid sequences of the specific recombinant carbonic anhydrase polypeptides included in the Sequence Listing of the present disclosure include an initiating methionine (M) residue (i.e., M represents residue position 1). The skilled artisan, however, understands that this initiating methionine residue can be removed by biological processing machinery, such as in a host cell or in vitro translation system, to generate a mature protein lacking the initiating methionine residue, but otherwise retaining the enzyme's properties. Consequently, the term “amino acid residue difference relative to SEQ ID NO:2 at position Xn” as used herein may refer to position “Xn” or to the corresponding position (e.g., position (X−1)n) in a reference sequence that has been processed so as to lack the starting methionine.

As used herein, the phrase “conservative amino acid substitutions” refers to the interchangeability of residues having similar side chains, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar defined class of amino acids. By way of example and not limitation, in some embodiments, an amino acid with an aliphatic side chain is substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with a hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acids having aromatic side chains is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is substituted with another amino acid with a basis side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and/or a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively. Exemplary conservative substitutions are provided in Table 1.

TABLE 1 Exemplary Conservative Amino Acid Substitutions Residue Potential Conservative Substitutions A, L, V, I Other aliphatic (A, L, V, I) Other non-polar (A, L, V, I, G, M) G, M Other non-polar (A, L, V, I, G, M) D, E Other acidic (D, E) K, R Other basic (K, R) N, Q, S,T Other polar H, Y, W, F Other aromatic (H, Y, W, F) C, P Non-polar

As used herein, the phrase “non-conservative substitution” refers to substitution of an amino acid in the polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affects (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine) (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of example and not limitation, an exemplary non-conservative substitution can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

As used herein, “deletion” refers to modification of the polypeptide by removal of one or more amino acids from the reference polypeptide. Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the polypeptide while retaining enzymatic activity and/or retaining the improved properties of an engineered enzyme. Deletions can be directed to the internal portions and/or terminal portions of the polypeptide. In various embodiments, the deletion can comprise a continuous segment or can be discontinuous.

As used herein, “insertion” refers to modification of the polypeptide by addition of one or more amino acids to the reference polypeptide. In some embodiments, the improved engineered PGA enzymes comprise insertions of one or more amino acids to the naturally occurring PGA polypeptide as well as insertions of one or more amino acids to engineered PGA polypeptides. Insertions can be in the internal portions of the polypeptide, or to the carboxy or amino terminus. Insertions as used herein include fusion proteins as is known in the art. The insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the naturally occurring polypeptide.

The term “amino acid substitution set” or “substitution set” refers to a group of amino acid substitutions in a polypeptide sequence, as compared to a reference sequence. A substitution set can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions. In some embodiments, a substitution set refers to the set of amino acid substitutions that is present in any of the variant PGAs listed in the Tables provided in the Examples.

As used herein, “fragment” refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence. Fragments can typically have about 80%, about 90%, about 95%, about 98%, or about 99% of the full-length PGA polypeptide, for example the polypeptide of SEQ ID NO:2. In some embodiments, the fragment is “biologically active” (i.e., it exhibits the same enzymatic activity as the full-length sequence).

As used herein, “isolated polypeptide” refers to a polypeptide that is substantially separated from other contaminants that naturally accompany it, e.g., protein, lipids, and polynucleotides. The term embraces polypeptides which have been removed or purified from their naturally-occurring environment or expression system (e.g., host cell or in vitro synthesis). The improved PGA enzymes may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations. As such, in some embodiments, the engineered PGA polypeptides of the present disclosure can be an isolated polypeptide.

As used herein, “substantially pure polypeptide” refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition), and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight. Generally, a substantially pure engineered PGA polypeptide composition comprises about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% of all macromolecular species by mole or % weight present in the composition. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated improved PGA polypeptide is a substantially pure polypeptide composition.

As used herein, when used in reference to a nucleic acid or polypeptide, the term “heterologous” refers to a sequence that is not normally expressed and secreted by an organism (e.g., a wild-type organism). In some embodiments, the term encompasses a sequence that comprises two or more subsequences which are not found in the same relationship to each other as normally found in nature, or is recombinantly engineered so that its level of expression, or physical relationship to other nucleic acids or other molecules in a cell, or structure, is not normally found in nature. For instance, a heterologous nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged in a manner not found in nature (e.g., a nucleic acid open reading frame (ORF) of the invention operatively linked to a promoter sequence inserted into an expression cassette, such as a vector). In some embodiments, “heterologous polynucleotide” refers to any polynucleotide that is introduced into a host cell by laboratory techniques, and includes polynucleotides that are removed from a host cell, subjected to laboratory manipulation, and then reintroduced into a host cell.

As used herein, “suitable reaction conditions” refer to those conditions in the biocatalytic reaction solution (e.g., ranges of enzyme loading, substrate loading, cofactor loading, temperature, pH, buffers, co-solvents, etc.) under which a PGA polypeptide of the present disclosure is capable of releasing free insulin by removing tri-phenyl acetate protecting groups. Exemplary “suitable reaction conditions” are provided in the present disclosure and illustrated by the Examples.

As used herein, “loading,” such as in “compound loading,” “enzyme loading,” or “cofactor loading” refers to the concentration or amount of a component in a reaction mixture at the start of the reaction.

As used herein, “substrate” in the context of a biocatalyst mediated process refers to the compound or molecule acted on by the biocatalyst.

As used herein “product” in the context of a biocatalyst mediated process refers to the compound or molecule resulting from the action of the biocatalyst.

As used herein, “equilibration” as used herein refers to the process resulting in a steady state concentration of chemical species in a chemical or enzymatic reaction (e.g., interconversion of two species A and B), including interconversion of stereoisomers, as determined by the forward rate constant and the reverse rate constant of the chemical or enzymatic reaction.

As used herein “acylase” and “acyltransferases” are used interchangeably to refer to enzymes that are capable of transferring an acyl group from a donor to an acceptor to form esters or amides. The acylase mediated reverse reaction results in hydrolysis of the ester or amide.

As used herein, “penicillin G” and “benzylpenicillin” refer to the antibiotic also known as (2S,5R,6R)-3,3-dimethyl-7-oxo-6-(2-phenylacetamido)-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid (C₁₆H₁₈N₂O₄S). It is primarily effective against Gram-positive organisms, although some Gram-negative organisms are also susceptible to it.

As used herein, “penicillin G acylase” and “PGA” are used interchangeably to refer to an enzyme having the capability of mediating cleavage of penicillin G (benzylpenicillin) to phenylacetic acid (PHA) and 6-aminopenicillanic acid (6-APA). In some embodiments, PGA activity can be based on cleavage of model substrates, for instance the cleavage of 6-nitro-3-(phenylacetamide)benzoic acid to phenylacetic acid and 5-amino-2-nitro-benzoic acid. PGAs are also capable of carrying out the reverse reaction of transferring an acyl group of an acyl donor to an acyl acceptor. PGAs as referred to herein, include naturally occurring (wild type) PGAs as well as non-naturally occurring PGA enzymes comprising one or more engineered polypeptides generated by human manipulation. The wild-type PGA gene is a heterodimer consisting of alpha subunit (23.8 KDa) and beta subunit (62.2KDa) linked by a spacer region of 54 amino acids. Due to the presence of the spacer region, an auto-processing step is required to form the active protein.

As used herein, “acyl donor” refers to that portion of the acylase substrate which donates the acyl group to an acyl acceptor to form esters or amides.

As used herein, “acyl acceptor” refers to that portion of the acylase substrate which accepts the acyl group of the acyl donor to form esters or amides.

As used herein, “α-chain sequence” refers to an amino acid sequence that corresponds to (e.g., has at least 85% identity to) the residues at positions 27 to 235 of SEQ ID NO: 2. As used herein, a single chain polypeptide can comprise an “α-chain sequence” and additional sequence(s).

As used herein, “β-chain sequence” refers to an amino acid sequence that corresponds to (e.g., has at least 85% identity to) residues at positions 290 to 846 of SEQ ID NO:2. As used herein, a single chain polypeptide can comprise a “β-chain sequence” and additional sequence(s).

As used herein, “derived from” when used in the context of engineered PGA enzymes, identifies the originating PGA enzyme, and/or the gene encoding such PGA enzyme, upon which the engineering was based. For example, the single chain engineered PGA enzyme of SEQ ID NO: 60 was obtained by artificially evolving, over multiple generations the gene encoding the K. citrophila PGA. Thus, in some embodiments, engineered PGA enzymes are derived from the naturally occurring or wild-type PGA of SEQ ID NO:2, while in some additional embodiments, the engineered PGA enzymes are derived from other evolved PGA enzymes. In some embodiments, the engineered PGA comprises an α-chain sequence and a β-chain sequence, which can be present as separate polypeptides in the mature enzyme, or be present as part of a single chain polypeptide. In some embodiments, when present as a single chain form, the engineered PGA polypeptide can comprise, from the amino to carboxy terminus, the structure

-   -   B-L-A         wherein B is the β-chain sequence (or B unit); A is the α-chain         sequence (or A unit); and L is a linker connecting the α-chain         to the β-chain sequences. In some embodiments, the spacer or         linker L comprises a spacer or linker of sufficient length and         flexibility to permit proper folding and interaction of the A         and B units to form a functional PGA enzyme. An exemplary         linker/space comprises the amino acid sequence Gln˜Leu˜Asp˜Gln.

Whether in the form of separate polypeptides or as a single chain polypeptide, the α- and β-chain sequences can have one or more residue differences as compared to the naturally occurring α- and β-chain sequences of K citrophila PGA.

As used herein, “insulin” refers to the polypeptide hormone produced by the beta-cells of the pancreas in normal individuals. Insulin is necessary for regulating carbohydrate metabolism, by reducing blood glucose levels. Systematic deficiency of insulin results in diabetes. Insulin is comprised of 51 amino acids and has a molecular weight of approximately 5800 daltons. Insulin is comprised of two peptide chains (designated “A” and “B”), containing one intrasubunit and two intersubunit disulfide bonds. The A chain is composed of 21 amino acids and the B chain is composed of 30 amino acids. The two chains form a highly ordered structure, with several alpha-helical regions in both the A and B chains. Isolated chains are inactive. In solution, insulin is either a monomer, dimer, or hexamer. It is hexameric in the highly concentrated preparations used for subcutaneous injection, but becomes monomeric as it is diluted in body fluids. The definition is intended to encompass proinsulin and any purified isolated polypeptide having part or all of the primary structural conformation and at least one of the biological properties of naturally-occurring insulin. It is further intended to encompass natural and synthetically-derived insulin, including glycoforms, as well as analogs (e.g., polypeptides having deletions, insertions, and/or substitutions).

Insulin contains three nucleophilic amines that can potentially react with a phenylacetate-donor and be deprotected by PGA. These residues include a Lys on the B-chain at position 29 (B29) and two N-terminal free amines, Gly on the A-chain at position 1 (A1) and Phe on the B-chain at position 1 (B1). Tri-protected insulin (phenyl acetate chemically attached to A1, B1, B29 residues on human insulin) is provided herein. PGA has previously been reported to catalyze hydrolysis of N-phenylacetate-protected peptides and insulin with exclusive selectivity for the phenylacetate amide bond, leaving the rest of the peptide bonds of the protein intact (Brtnik et al., Coll. Czech. Chem. Commun., 46 (8), 1983-1989 [1981]; and Wang et al. Biopolym. 25 (Suppl.), S109-S114 [1986]).

As used herein, “tri-phenyl acetate protecting group,” refers to an insulin molecule that has the three primary amines at the B1, B29 and A1 positions that are protected with a phenyl acyl group.

As used herein, “di-phenyl acetate protecting group” refers to an insulin molecule that has the two primary amines at the B1, B29 and/or the A1 positions that are protected with a phenyl acyl group.

As used herein, “di-phenyl acetate protecting group” refers to an insulin molecule that has one primary amine at the B1, B29 or the A1 positions that are protected with a phenyl acyl group.

Penicillin G Acylases

Penicillin acylase was first described from Penicillium chrysogenum Wisc. Q176 by Sakaguchi and Murao (Sakaguchi and Murao, J. Agr. Chem. Soc. Jpn., 23:411 [1950]). Penicillin G acylase is a hydrolytic enzyme that acts on the side chains of penicillin G, cephalosporin G, and related antibiotics to produce the β-lactam antibiotic intermediates 6-amino penicillanic acid and 7-amino des-acetoxy cephalosporanic acid, with phenyl acetic acid as a common by-product. These antibiotic intermediates are among the potential building blocks of semi-synthetic antibiotics, such as ampicillin, amoxicillin, cloxacillin, cephalexin, and cefatoxime.

As indicated above, penicillin G acylases (PGA) are characterized by the ability to catalyze the hydrolytic cleavage of penicillin G, with a conjugate base of structural formula (I), to 6-amino penicillanic acid, with a conjugate base of structural formula (II), and phenylacetic acid of structural formula (III), as shown in Scheme 1:

While not being bound by theory, substrate specificity appears associated with recognition of the hydrophobic phenyl group while a nucleophile, which in some PGAs is a serine residue at the N-terminus of the beta-chain acts as the acceptor of beta-lactam and a variety of other groups, such as beta-amino acids. PGAs can also be characterized by the ability to cleave a model substrates analogous to penicillin G, for instance cleavage of 6-nitro-3-(phenylacetamido)benzoic acid (NIPAB) of structural formula (IV), as shown in Scheme 2:

to phenylacetic acid of structural formula (III) and 5-amino-2-nitro-benzoic acid of structural formula (V) (See e.g., Alkema et al., Anal. Biochem., 275:47-53 [1999]). Because the 5-amino-2-nitro-benzoic acid is chromogenic, the substrate of formula (IV) provides a convenient way of measuring PGA activity. In addition to the foregoing reactions, PGAs can also be used in the kinetic resolution of DL-tert leucine for the preparation of optically pure tert leucine (See e.g., Liu et al., Prep. Biochem. Biotechnol., 36:235-41 [2006]).

The PGAs of the present invention are based on the enzyme obtained from the organism Kluyvera citrophila (K. citrophila). As with PGAs from other organisms, the PGA of K. citrophila is a heterodimeric enzyme comprised of an alpha-subunit and a beta-subunit that is generated by proteolytic processing of a pre-pro-PGA polypeptide. Removal of a signal peptide and a spacer peptide produces the mature heterodimer (See e.g., Barbero et al., Gene 49:69-80 [1986]). The amino acid sequence of the naturally occurring pre-pro-PGA polypeptide of K. citrophila is publicly available (See e.g., Genbank accession No. P07941, [gi:129551]) and is provided herein as SEQ ID NO:2. The alpha-chain sequence of the naturally occurring K. citrophila PGA corresponds to residues 27 to 235 of SEQ ID NO:2. The beta-chain sequence of the naturally occurring K. citrophila PGA corresponds to residues 290 to 846 of SEQ ID NO:2. Residues 1 to 26 of SEQ ID NO:2 correspond to the signal peptide and residues 236-289 of SEQ ID NO:2 correspond to the linking propeptide, both of which are removed to generate the naturally occurring mature PGA enzyme which is a heterodimer comprising an α-chain subunit and a 3-chain subunit. In some embodiments, the engineered PGA comprises an α-chain sequence and a β-chain sequence, which can be present as separate polypeptides in the mature enzyme, or be present as part of a single chain polypeptide. In some embodiments, when present as a single chain form, the engineered PGA polypeptide can comprise, from the amino to carboxy terminus, the structure

-   -   B-L-A         wherein B is the β-chain sequence (or B unit); A is the α-chain         sequence (or A unit); and L is a linker connecting the α-chain         to the β-chain sequences. In some embodiments, the spacer or         linker L comprises a spacer or linker of sufficient length and         flexibility to permit proper folding and interaction of the A         and B units to form a functional PGA enzyme. An exemplary         linker/space comprises the amino acid sequence Gln˜Leu˜Asp˜Gln.

Whether in the form of separate polypeptides or as a single chain polypeptide, the α- and β-chain sequences can have one or more residue differences as compared to the naturally occurring α- and β-chain sequences of K. citrophila PGA.

In some embodiments, the present invention provides engineered PGA polypeptides with amino acid sequences that have at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NOS:12, 24, 40, 56, 70, 82, 100, 108, 110, 116, 136, 154, and/or 160.

The present invention provides insulin-specific acylation biocatalysts suitable for commercial scale use. Directed evolution was used to develop efficient acylase variants capable of adding the phenyl acetate protecting group to insulin at the A1, B1, and/or B29 positions. The PGA variants provided herein are capable of accepting a wide range of acyl groups, exhibit increased solvent stability, and improved thermostability, as compared to the wild-type PGA. The variant PGAs provided herein lack the spacer region. Thus, the auto-processing step is not required in order to produce active enzymes. The present invention also provides polynucleotides encoding the engineered PGA polypeptides. In some embodiments, the polynucleotides are operatively linked to one or more heterologous regulatory sequences that control gene expression, to create a recombinant polynucleotide capable of expressing the polypeptide. Expression constructs containing a heterologous polynucleotide encoding the engineered PGA polypeptides can be introduced into appropriate host cells to express the corresponding PGA polypeptide. Because of the knowledge of the codons corresponding to the various amino acids, availability of a protein sequence provides a description of all the polynucleotides capable of encoding the subject. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons allows an extremely large number of nucleic acids to be made, all of which encode the improved PGA enzymes disclosed herein. Thus, having identified a particular amino acid sequence, those skilled in the art could make any number of different nucleic acids by simply modifying the sequence of one or more codons in a way which does not change the amino acid sequence of the protein. In this regard, the present disclosure specifically contemplates each and every possible variation of polynucleotides that could be made by selecting combinations based on the possible codon choices, and all such variations are to be considered specifically disclosed for any polypeptide disclosed herein, including the amino acid sequences presented in the Tables in the Examples.

In various embodiments, the codons are preferably selected to fit the host cell in which the protein is being produced. For example, preferred codons used in bacteria are used to express the gene in bacteria; preferred codons used in yeast are used for expression in yeast; and preferred codons used in mammals are used for expression in mammalian cells.

In certain embodiments, all codons need not be replaced to optimize the codon usage of the PGA polypeptides since the natural sequence will comprise preferred codons and because use of preferred codons may not be required for all amino acid residues. Consequently, codon optimized polynucleotides encoding the PGA enzymes may contain preferred codons at about 40%, 50%, 60%, 70%, 80%, or greater than 90% of codon positions of the full length coding region.

In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a PGA polypeptide with an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the alpha-chain and/or beta-chain any of the reference engineered PGA polypeptides described herein. Accordingly, in some embodiments, the polynucleotide encodes an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a reference alpha- and beta-chain sequences based on SEQ ID NO: 12, 24, 40, 56, 70, 82, 100, 108, 110, 116, 136, 154, and/or 160. In some embodiments, the polynucleotide encodes an alpha- and/or beta-chain amino acid sequence of SEQ ID NO: 12, 24, 40, 56, 70, 82, 100, 108, 110, 116, 136, 154, and/or 160.

In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a PGA polypeptide with an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 12, 24, 40, 56, 70, 82, 100, 108, 110, 116, 136, 154, and/or 160. Accordingly, in some embodiments, the polynucleotide encodes an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO12, 24, 40, 56, 70, 82, 100, 108, 110, 116, 136, 154, and/or 160.

In some embodiments, an isolated polynucleotide encoding an improved PGA polypeptide was manipulated in a variety of ways to provide for improved activity and/or expression of the polypeptide. Manipulation of the isolated polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are well known in the art.

For example, mutagenesis and directed evolution methods can be readily applied to polynucleotides to generate variant libraries that can be expressed, screened, and assayed. Mutagenesis and directed evolution methods are well known in the art (See e.g., U.S. Pat. Nos. 5,605,793, 5,830,721, 6,132,970, 6,420,175, 6,277,638, 6,365,408, 6,602,986, 7,288,375, 6,287,861, 6,297,053, 6,576,467, 6,444,468, 5,811238, 6,117,679, 6,165,793, 6,180,406, 6,291,242, 6,995,017, 6,395,547, 6,506,602, 6,519,065, 6,506,603, 6,413,774, 6,573,098, 6,323,030, 6,344,356, 6,372,497, 7,868,138, 5,834,252, 5,928,905, 6,489,146, 6,096,548, 6,387,702, 6,391,552, 6,358,742, 6,482,647, 6,335,160, 6,653,072, 6,355,484, 6,03,344, 6,319,713, 6,613,514, 6,455,253, 6,579,678, 6,586,182, 6,406,855, 6,946,296, 7,534,564, 7,776,598, 5,837,458, 6,391,640, 6,309,883, 7,105,297, 7,795,030, 6,326,204, 6,251,674, 6,716,631, 6,528,311, 6,287,862, 6,335,198, 6,352,859, 6,379,964, 7,148,054, 7,629,170, 7,620,500, 6,365,377, 6,358,740, 6,406,910, 6,413,745, 6,436,675, 6,961,664, 7,430,477, 7,873,499, 7,702,464, 7,783,428, 7,747,391, 7,747,393, 7,751,986, 6,376,246, 6,426,224, 6,423,542, 6,479,652, 6,319,714, 6,521,453, 6,368,861, 7,421,347, 7,058,515, 7,024,312, 7,620,502, 7,853,410, 7,957,912, 7,904,249, and all related non-US counterparts; Ling et al., Anal. Biochem., 254(2):157-78 [1997]; Dale et al., Meth. Mol. Biol., 57:369-74 [1996]; Smith, Ann. Rev. Genet., 19:423-462 [1985]; Botstein et al., Science, 229:1193-1201 [1985]; Carter, Biochem. J., 237:1-7 [1986]; Kramer et al., Cell, 38:879-887 [1984]; Wells et al., Gene, 34:315-323 [1985]; Minshull et al., Curr. Op. Chem. Biol., 3:284-290 [1999]; Christians et al., Nat. Biotechnol., 17:259-264 [1999]; Crameri et al., Nature, 391:288-291 [1998]; Crameri, et al., Nat. Biotechnol., 15:436-438 [1997]; Zhang et al., Proc. Nat. Acad. Sci. U.S.A., 94:4504-4509 [1997]; Crameri et al., Nat. Biotechnol., 14:315-319 [1996]; Stemmer, Nature, 370:389-391 [1994]; Stemmer, Proc. Nat. Acad. Sci. USA, 91:10747-10751 [1994]; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767; and WO 2009/152336, all of which are incorporated herein by reference).

In some embodiments, the variant PGA acylases of the present invention further comprise additional sequences that do not alter the encoded activity of the enzyme. For example, in some embodiments, the variant PGA acylases are linked to an epitope tag or to another sequence useful in purification.

In some embodiments, the variant PGA acylase polypeptides of the present invention are secreted from the host cell in which they are expressed (e.g., a yeast or filamentous fungal host cell) and are expressed as a pre-protein including a signal peptide (i.e., an amino acid sequence linked to the amino terminus of a polypeptide and which directs the encoded polypeptide into the cell secretory pathway).

In some embodiments, the signal peptide is an endogenous K. citrophila PGA acylase signal peptide. In some other embodiments, signal peptides from other K, citrophila secreted proteins are used.

In some embodiments, other signal peptides find use, depending on the host cell and other factors. Effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to, the signal peptide coding regions obtained from Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, Humicola lanuginosa lipase, and T. reesei cellobiohydrolase II. Signal peptide coding regions for bacterial host cells include, but are not limited to the signal peptide coding regions obtained from the genes for Bacillus NCB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. In some additional embodiments, other signal peptides find use in the present invention (See e.g., Simonen and Palva, Microbiol. Rev., 57: 109-137 [1993], incorporated herein by reference). Additional useful signal peptides for yeast host cells include those from the genes for Saccharomyces cerevisiae alpha-factor, Saccharomyces cerevisiae SUC2 invertase (See e.g., Taussig and Carlson, Nucl. Acids Res., 11:1943-54 [1983]; SwissProt Accession No. P00724; and Romanos et al., Yeast 8:423-488 [1992]). In some embodiments, variants of these signal peptides and other signal peptides find use. Indeed, it is not intended that the present invention be limited to any specific signal peptide, as any suitable signal peptide known in the art finds use in the present invention.

In some embodiments, the present invention provides polynucleotides encoding variant PGA acylase polypeptides, and/or biologically active fragments thereof, as described herein. In some embodiments, the polynucleotide is operably linked to one or more heterologous regulatory or control sequences that control gene expression to create a recombinant polynucleotide capable of expressing the polypeptide. In some embodiments, expression constructs containing a heterologous polynucleotide encoding a variant PGA acylase is introduced into appropriate host cells to express the variant PGA acylase.

Those of ordinary skill in the art understand that due to the degeneracy of the genetic code, a multitude of nucleotide sequences encoding variant PGA acylase polypeptides of the present invention exist. For example, the codons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine. Thus, at every position in the nucleic acids of the invention where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described above without altering the encoded polypeptide. It is understood that “U” in an RNA sequence corresponds to “T” in a DNA sequence. The invention contemplates and provides each and every possible variation of nucleic acid sequence encoding a polypeptide of the invention that could be made by selecting combinations based on possible codon choices.

As indicated above, DNA sequence encoding a PGA may also be designed for high codon usage bias codons (codons that are used at higher frequency in the protein coding regions than other codons that code for the same amino acid). The preferred codons may be determined in relation to codon usage in a single gene, a set of genes of common function or origin, highly expressed genes, the codon frequency in the aggregate protein coding regions of the whole organism, codon frequency in the aggregate protein coding regions of related organisms, or combinations thereof. A codon whose frequency increases with the level of gene expression is typically an optimal codon for expression. In particular, a DNA sequence can be optimized for expression in a particular host organism. A variety of methods are well-known in the art for determining the codon frequency (e.g., codon usage, relative synonymous codon usage) and codon preference in specific organisms, including multivariate analysis (e.g., using cluster analysis or correspondence analysis,) and the effective number of codons used in a gene. The data source for obtaining codon usage may rely on any available nucleotide sequence capable of coding for a protein.

These data sets include nucleic acid sequences actually known to encode expressed proteins (e.g., complete protein coding sequences-CDS), expressed sequence tags (ESTs), or predicted coding regions of genomic sequences, as is well-known in the art. Polynucleotides encoding variant PGAs can be prepared using any suitable methods known in the art. Typically, oligonucleotides are individually synthesized, then joined (e.g., by enzymatic or chemical ligation methods, or polymerase-mediated methods) to form essentially any desired continuous sequence. In some embodiments, polynucleotides of the present invention are prepared by chemical synthesis using, any suitable methods known in the art, including but not limited to automated synthetic methods. For example, in the phosphoramidite method, oligonucleotides are synthesized (e.g., in an automatic DNA synthesizer), purified, annealed, ligated and cloned in appropriate vectors. In some embodiments, double stranded DNA fragments are then obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence. There are numerous general and standard texts that provide methods useful in the present invention are well known to those skilled in the art.

The engineered PGAs can be obtained by subjecting the polynucleotide encoding the naturally occurring PGA to mutagenesis and/or directed evolution methods, as discussed above. Mutagenesis may be performed in accordance with any of the techniques known in the art, including random and site-specific mutagenesis. Directed evolution can be performed with any of the techniques known in the art to screen for improved variants including shuffling. Other directed evolution procedures that find use include, but are not limited to staggered extension process (StEP), in vitro recombination, mutagenic PCR, cassette mutagenesis, splicing by overlap extension (SOEing), ProSAR™ directed evolution methods, etc., as well as any other suitable methods.

The clones obtained following mutagenesis treatment are screened for engineered PGAs having a desired improved enzyme property. Measuring enzyme activity from the expression libraries can be performed using the standard biochemistry technique of monitoring the rate of product formation. Where an improved enzyme property desired is thermal stability, enzyme activity may be measured after subjecting the enzyme preparations to a defined temperature and measuring the amount of enzyme activity remaining after heat treatments. Clones containing a polynucleotide encoding a PGA are then isolated, sequenced to identify the nucleotide sequence changes (if any), and used to express the enzyme in a host cell.

When the sequence of the engineered polypeptide is known, the polynucleotides encoding the enzyme can be prepared by standard solid-phase methods, according to known synthetic methods. In some embodiments, fragments of up to about 100 bases can be individually synthesized, then joined (e.g., by enzymatic or chemical ligation methods, or polymerase mediated methods) to form any desired continuous sequence. For example, polynucleotides and oligonucleotides of the invention can be prepared by chemical synthesis (e.g., using the classical phosphoramidite method described by Beaucage et al., Tet. Lett., 22:1859-69 [1981], or the method described by Matthes et al., EMBO J., 3:801-05 [1984], as it is typically practiced in automated synthetic methods). According to the phosphoramidite method, oligonucleotides are synthesized (e.g., in an automatic DNA synthesizer), purified, annealed, ligated and cloned in appropriate vectors. In addition, essentially any nucleic acid can be obtained from any of a variety of commercial sources (e.g., The Midland Certified Reagent Company, Midland, Tex., The Great American Gene Company, Ramona, Calif., ExpressGen Inc. Chicago, Ill., Operon Technologies Inc., Alameda, Calif., and many others).

The present invention also provides recombinant constructs comprising a sequence encoding at least one variant PGA, as provided herein. In some embodiments, the present invention provides an expression vector comprising a variant PGA polynucleotide operably linked to a heterologous promoter. In some embodiments, expression vectors of the present invention are used to transform appropriate host cells to permit the host cells to express the variant PGA protein. Methods for recombinant expression of proteins in fungi and other organisms are well known in the art, and a number of expression vectors are available or can be constructed using routine methods. In some embodiments, nucleic acid constructs of the present invention comprise a vector, such as, a plasmid, a cosmid, a phage, a virus, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), and the like, into which a nucleic acid sequence of the invention has been inserted. In some embodiments, polynucleotides of the present invention are incorporated into any one of a variety of expression vectors suitable for expressing variant PGA polypeptide(s). Suitable vectors include, but are not limited to chromosomal, nonchromosomal and synthetic DNA sequences (e.g., derivatives of SV40), as well as bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, pseudorabies, adenovirus, adeno-associated virus, retroviruses, and many others. Any suitable vector that transduces genetic material into a cell, and, if replication is desired, which is replicable and viable in the relevant host finds use in the present invention.

In some embodiments, the construct further comprises regulatory sequences, including but not limited to a promoter, operably linked to the protein encoding sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art. Indeed, in some embodiments, in order to obtain high levels of expression in a particular host it is often useful to express the variant PGAs of the present invention under the control of a heterologous promoter. In some embodiments, a promoter sequence is operably linked to the 5′ region of the variant PGA coding sequence using any suitable method known in the art. Examples of useful promoters for expression of variant PGAs include, but are not limited to promoters from fungi. In some embodiments, a promoter sequence that drives expression of a gene other than a PGA gene in a fungal strain finds use. As a non-limiting example, a fungal promoter from a gene encoding an endoglucanase may be used. In some embodiments, a promoter sequence that drives the expression of a PGA gene in a fungal strain other than the fungal strain from which the PGAs were derived finds use. Examples of other suitable promoters useful for directing the transcription of the nucleotide constructs of the present invention in a filamentous fungal host cell include, but are not limited to promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (See e.g., WO 96/00787, incorporated herein by reference), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), promoters such as cbh1, cbh2, egl1, egl2, pepA, hfb1, hfb2, xynl, amy, and glaA (See e.g., Nunberg et al., Mol. Cell Biol., 4:2306-2315 [1984]; Boel et al., EMBO J., 3:1581-85 [1984]; and European Patent Appln. 137280, all of which are incorporated herein by reference), and mutant, truncated, and hybrid promoters thereof.

In yeast host cells, useful promoters include, but are not limited to those from the genes for Saccharomyces cerevisiae enolase (eno-1), Saccharomyces cerevisiae galactokinase (gall), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), and S. cerevisiae 3-phosphoglycerate kinase. Additional useful promoters useful for yeast host cells are known in the art (See e.g., Romanos et al., Yeast 8:423-488 [1992], incorporated herein by reference). In addition, promoters associated with chitinase production in fungi find use in the present invention (See e.g., Blaiseau and Lafay, Gene 120243-248 [1992]; and Limon et al., Curr. Genet., 28:478-83 [1995], both of which are incorporated herein by reference).

For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure, include but are not limited to the promoters obtained from the E. coli lac operon, E. coli trp operon, bacteriophage X, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (See e.g., Villa-Kamaroff et al., Proc. Natl. Acad. Sci. USA 75: 3727-3731 [1978]), as well as the tac promoter (See e.g., DeBoer et al., Proc. Natl. Acad. Sci. USA 80: 21-25 [1983]).

In some embodiments, cloned variant PGAs of the present invention also have a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3′ terminus of the nucleic acid sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice finds use in the present invention. Exemplary transcription terminators for filamentous fungal host cells include, but are not limited to those obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease (See also, U.S. Pat. No. 7,399,627, incorporated herein by reference). In some embodiments, exemplary terminators for yeast host cells include those obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYCl), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are well-known to those skilled in the art (See e.g., Romanos et al., Yeast 8:423-88 [1992]).

In some embodiments, a suitable leader sequence is part of a cloned variant PGA sequence, which is a nontranslated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5′ terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice finds use in the present invention. Exemplary leaders for filamentous fungal host cells include, but are not limited to those obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells include, but are not limited to those obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

In some embodiments, the sequences of the present invention also comprise a polyadenylation sequence, which is a sequence operably linked to the 3′ terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the host cell of choice finds use in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to those obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are known in the art (See e.g., Guo and Sherman, Mol. Cell. Biol., 15:5983-5990 [1995]).

In some embodiments, the control sequence comprises a signal peptide coding region encoding an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway. The 5′ end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, the 5′ end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. The foreign signal peptide coding region may be required where the coding sequence does not naturally contain a signal peptide coding region.

Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the polypeptide. However, any signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of choice may be used in the present invention.

Effective signal peptide coding regions for bacterial host cells include, but are not limited to the signal peptide coding regions obtained from the genes for Bacillus NCB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are known in the art (See e.g., Simonen and Palva, Microbiol. Rev., 57: 109-137 [1993]).

Effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase.

Useful signal peptides for yeast host cells include, but are not limited to genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding regions are known in the art (See e.g., Romanos et al., [1992], supra).

In some embodiments, the control sequence comprises a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to a mature active PGA polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila lactase (See e.g., WO 95/33836).

Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region.

In some embodiments, regulatory sequences are also used to allow the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those which cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to the lac, tac, and trp operator systems. In yeast host cells, suitable regulatory systems include, as examples, the ADH2 system or GAL1 system. In filamentous fungi, suitable regulatory sequences include the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter.

Other examples of regulatory sequences are those which allow for gene amplification. In eukaryotic systems, these include the dihydrofolate reductase gene, which is amplified in the presence of methotrexate, and the metallothionein genes, which are amplified with heavy metals. In these cases, the nucleic acid sequence encoding the PGA polypeptide of the present invention would be operably linked with the regulatory sequence.

Thus, in additional embodiments, the present invention provides recombinant expression vectors comprising a polynucleotide encoding an engineered PGA polypeptide or a variant thereof, and one or more expression regulating regions such as a promoter and a terminator, a replication origin, etc., depending on the type of hosts into which they are to be introduced. In some embodiments, the various nucleic acid and control sequences described above are joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the polypeptide at such sites. Alternatively, in some embodiments, the nucleic acid sequences are expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

The recombinant expression vector comprises any suitable vector (e.g., a plasmid or virus), that can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the polynucleotide sequence. The choice of the vector typically depends on the compatibility of the vector with the host cell into which the vector is to be introduced. In some embodiments, the vectors are linear or closed circular plasmids.

In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome). In some embodiments, the vector contains any means for assuring self-replication. Alternatively, in some other embodiments, upon being introduced into the host cell, the vector is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, in additional embodiments, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon find use.

In some embodiments, the expression vector of the present invention contains one or more selectable markers, which permit easy selection of transformed cells. A “selectable marker” is a gene, the product of which provides for biocide or viral resistance, resistance to antimicrobials or heavy metals, prototrophy to auxotrophs, and the like. Any suitable selectable markers for use in a filamentous fungal host cell find use in the present invention, including, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Additional markers useful in host cells such as Aspergillus, include but are not limited to the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae, and the bar gene of Streptomyces hygroscopicus. Suitable markers for yeast host cells include, but are not limited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Examples of bacterial selectable markers include, but are not limited to the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers, which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, and or tetracycline resistance.

In some embodiments, the expression vectors of the present invention contain an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome. In some embodiments involving integration into the host cell genome, the vectors rely on the nucleic acid sequence encoding the polypeptide or any other element of the vector for integration of the vector into the genome by homologous or nonhomologous recombination.

In some alternative embodiments, the expression vectors contain additional nucleic acid sequences for directing integration by homologous recombination into the genome of the host cell. The additional nucleic acid sequences enable the vector to be integrated into the host cell genome at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements preferably contain a sufficient number of nucleotides, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleic acid sequences. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. Examples of bacterial origins of replication are P15A on or the origins of replication of plasmids pBR322, pUC19, pACYC177 (which plasmid has the P15A ori), or pACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060, or pAMP1 permitting replication in Bacillus. Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6. The origin of replication may be one having a mutation which makes its functioning temperature-sensitive in the host cell (See e.g., Ehrlich, Proc. Natl. Acad. Sci. USA 75:1433 [1978]).

In some embodiments, more than one copy of a nucleic acid sequence of the present invention is inserted into the host cell to increase production of the gene product. An increase in the copy number of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the nucleic acid sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleic acid sequence, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

Many of the expression vectors for use in the present invention are commercially available. Suitable commercial expression vectors include, but are not limited to the p3xFLAG™™ expression vectors (Sigma-Aldrich Chemicals), which include a CMV promoter and hGH polyadenylation site for expression in mammalian host cells and a pBR322 origin of replication and ampicillin resistance markers for amplification in E. coli. Other suitable expression vectors include, but are not limited to pBluescriptII SK(−) and pBK-CMV (Stratagene), and plasmids derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen) or pPoly (See e.g., Lathe et al., Gene 57:193-201 [1987]).

Thus, in some embodiments, a vector comprising a sequence encoding at least one variant PGA is transformed into a host cell in order to allow propagation of the vector and expression of the variant PGA(s). In some embodiments, the variant PGAs are post-translationally modified to remove the signal peptide, and in some cases may be cleaved after secretion. In some embodiments, the transformed host cell described above is cultured in a suitable nutrient medium under conditions permitting the expression of the variant PGA(s). Any suitable medium useful for culturing the host cells finds use in the present invention, including, but not limited to minimal or complex media containing appropriate supplements. In some embodiments, host cells are grown in HTP media. Suitable media are available from various commercial suppliers or may be prepared according to published recipes (e.g., in catalogues of the American Type Culture Collection).

In another aspect, the present invention provides host cells comprising a polynucleotide encoding an improved PGA polypeptide provided herein, the polynucleotide being operatively linked to one or more control sequences for expression of the PGA enzyme in the host cell. Host cells for use in expressing the PGA polypeptides encoded by the expression vectors of the present invention are well known in the art and include but are not limited to, bacterial cells, such as E. coli, Bacillus megaterium, Lactobacillus kefir, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells. Appropriate culture media and growth conditions for the above-described host cells are well known in the art.

Polynucleotides for expression of the PGA may be introduced into cells by various methods known in the art. Techniques include among others, electroporation, biolistic particle bombardment, liposome mediated transfection, calcium chloride transfection, and protoplast fusion. Various methods for introducing polynucleotides into cells are known to those skilled in the art.

In some embodiments, the host cell is a eukaryotic cell. Suitable eukaryotic host cells include, but are not limited to, fungal cells, algal cells, insect cells, and plant cells. Suitable fungal host cells include, but are not limited to, Ascomycota, Basidiomycota, Deuteromycota, Zygomycota, Fungi imperfecti. In some embodiments, the fungal host cells are yeast cells and filamentous fungal cells. The filamentous fungal host cells of the present invention include all filamentous forms of the subdivision Eumycotina and Oomycota. Filamentous fungi are characterized by a vegetative mycelium with a cell wall composed of chitin, cellulose and other complex polysaccharides. The filamentous fungal host cells of the present invention are morphologically distinct from yeast.

In some embodiments of the present invention, the filamentous fungal host cells are of any suitable genus and species, including, but not limited to Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus, Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora, Mucor, Neurospora, Penicillium, Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia, Trametes, Tolypocladium, Trichoderma, Verticillium, and/or Volvariella, and/or teleomorphs, or anamorphs, and synonyms, basionyms, or taxonomic equivalents thereof.

In some embodiments of the present invention, the host cell is a yeast cell, including but not limited to cells of Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, or Yarrowia species. In some embodiments of the present invention, the yeast cell is Hansenula polymorpha, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis, Saccharomyces kluyveri, Schizosaccharomyces pombe, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia kodamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia quercuum, Pichia pijperi, Pichia stipitis, Pichia methanolica, Pichia angusta, Kluyveromyces lactis, Candida albicans, or Yarrowia lipolytica.

In some embodiments of the invention, the host cell is an algal cell such as Chlamydomonas (e.g., C. reinhardtii) and Phormidium (P. sp. ATCC29409).

In some other embodiments, the host cell is a prokaryotic cell. Suitable prokaryotic cells include, but are not limited to Gram-positive, Gram-negative and Gram-variable bacterial cells. Any suitable bacterial organism finds use in the present invention, including but not limited to Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter, Acidothermus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium, Brevibacterium, Butyrivibrio, Buchnera, Campestris, Camplyobacter, Clostridium, Corynebacterium, Chromatium, Coprococcus, Escherichia, Enterococcus, Enterobacter, Erwinia, Fusobacterium, Faecalibacterium, Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Ilyobacter, Micrococcus, Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium, Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus, Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia, Rhodospirillum, Rhodococcus, Scenedesmus, Streptomyces, Streptococcus, Synecoccus, Saccharomonospora, Staphylococcus, Serratia, Salmonella, Shigella, Thermoanaerobacterium, Tropheryma, Tularensis, Temecula, Thermosynechococcus, Thermococcus, Ureaplasma, Xanthomonas, Xylella, Yersinia and Zymomonas. In some embodiments, the host cell is a species of Agrobacterium, Acinetobacter, Azobacter, Bacillus, Bifidobacterium, Buchnera, Geobacillus, Campylobacter, Clostridium, Corynebacterium, Escherichia, Enterococcus, Erwinia, Flavobacterium, Lactobacillus, Lactococcus, Pantoea, Pseudomonas, Staphylococcus, Salmonella, Streptococcus, Streptomyces, or Zymomonas. In some embodiments, the bacterial host strain is non-pathogenic to humans. In some embodiments the bacterial host strain is an industrial strain. Numerous bacterial industrial strains are known and suitable in the present invention. In some embodiments of the present invention, the bacterial host cell is an Agrobacterium species (e.g., A. radiobacter, A. rhizogenes, and A. rubi). In some embodiments of the present invention, the bacterial host cell is an Arthrobacter species (e.g., A. aurescens, A. citreus, A. globiformis, A. hydrocarboglutamicus, A. mysorens, A. nicotianae, A. paraffineus, A. protophonniae, A. roseoparqffinus, A. sulfureus, and A. ureafaciens). In some embodiments of the present invention, the bacterial host cell is a Bacillus species (e.g., B. thuringensis, B. anthracis, B. megaterium, B. subtilis, B. lentus, B. circulans, B. pumilus, B. lautus, B.coagulans, B. brevis, B. firmus, B. alkaophius, B. licheniformis, B. clausii, B. stearothermophilus, B. halodurans, and B. amyloliquefaciens). In some embodiments, the host cell is an industrial Bacillus strain including but not limited to B. subtilis, B. pumilus, B. licheniformis, B. megaterium, B. clausii, B. stearothermophilus, or B. amyloliquefaciens. In some embodiments, the Bacillus host cells are B. subtilis, B. licheniformis, B. megaterium, B. stearothermophilus, and/or B. amyloliquefaciens. In some embodiments, the bacterial host cell is a Clostridium species (e.g., C. acetobutylicum, C. tetani E88, C. lituseburense, C. saccharobutylicum, C. perfringens, and C. beijerinckii). In some embodiments, the bacterial host cell is a Corynebacterium species (e.g., C. glutamicum and C. acetoacidophilum). In some embodiments the bacterial host cell is an Escherichia species (e.g., E. coli). In some embodiments, the bacterial host cell is an Erwinia species (e.g., E. uredovora, E. carotovora, E. ananas, E. herbicola, E. punctata, and E. terreus). In some embodiments, the bacterial host cell is a Pantoea species (e.g., P. citrea, and P. agglomerans). In some embodiments the bacterial host cell is a Pseudomonas species (e.g., P. putida, P. aeruginosa, P. mevalonii, and P. sp. D-01 10). In some embodiments, the bacterial host cell is a Streptococcus species (e.g., S. equisimiles, S. pyogenes, and S. uberis). In some embodiments, the bacterial host cell is a Streptomyces species (e.g., S. ambofaciens, S. achromogenes, S. avermitilis, S. coelicolor, S. aureofaciens, S. aureus, S.fungicidicus, S. griseus, and S. lividans). In some embodiments, the bacterial host cell is a Zymomonas species (e.g., Z. mobilis, and Z. lipolytica).

An exemplary host cell is Escherichia coli W3110. The expression vector was created by operatively linking a polynucleotide encoding an improved PGA into the plasmid pCK110900 operatively linked to the lac promoter under control of the lacI repressor. The expression vector also contained the P15a origin of replication and the chloramphenicol resistance gene. Cells containing the subject polynucleotide in Escherichia coli W3110 were isolated by subjecting the cells to chloramphenicol selection.

Many prokaryotic and eukaryotic strains that find use in the present invention are readily available to the public from a number of culture collections such as American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

In some embodiments, host cells are genetically modified to have characteristics that improve protein secretion, protein stability and/or other properties desirable for expression and/or secretion of a protein. Genetic modification can be achieved by genetic engineering techniques and/or classical microbiological techniques (e.g., chemical or UV mutagenesis and subsequent selection). Indeed, in some embodiments, combinations of recombinant modification and classical selection techniques are used to produce the host cells. Using recombinant technology, nucleic acid molecules can be introduced, deleted, inhibited or modified, in a manner that results in increased yields of PGA variant(s) within the host cell and/or in the culture medium. For example, knockout of Alp1 function results in a cell that is protease deficient, and knockout of pyr5 function results in a cell with a pyrimidine deficient phenotype. In one genetic engineering approach, homologous recombination is used to induce targeted gene modifications by specifically targeting a gene in vivo to suppress expression of the encoded protein. In alternative approaches, siRNA, antisense and/or ribozyme technology find use in inhibiting gene expression. A variety of methods are known in the art for reducing expression of protein in cells, including, but not limited to deletion of all or part of the gene encoding the protein and site-specific mutagenesis to disrupt expression or activity of the gene product. (See e.g., Chaveroche et al., Nucl. Acids Res., 28:22 e97 [2000]; Cho et al., Molec. Plant Microbe Interact., 19:7-15 [2006]; Maruyama and Kitamoto, Biotechnol Lett., 30:1811-1817 [2008]; Takahashi et al., Mol. Gen. Genom., 272: 344-352 [2004]; and You et al; Arch. Micriobiol.,191:615-622 [2009], all of which are incorporated by reference herein). Random mutagenesis, followed by screening for desired mutations also finds use (See e.g., Combier et al., FEMS Microbiol. Lett., 220:141-8 [2003]; and Firon et al., Eukary. Cell 2:247-55 [2003], both of which are incorporated by reference).

Introduction of a vector or DNA construct into a host cell can be accomplished using any suitable method known in the art, including but not limited to calcium phosphate transfection, DEAE-Dextran mediated transfection, PEG-mediated transformation, electroporation, or other common techniques known in the art.

In some embodiments, the engineered host cells (i.e., “recombinant host cells”) of the present invention are cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the PGA polynucleotide. Culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and are well-known to those skilled in the art. As noted, many standard references and texts are available for the culture and production of many cells, including cells of bacterial, plant, animal (especially mammalian) and archebacterial origin.

In some embodiments, cells expressing the variant PGA polypeptides of the invention are grown under batch or continuous fermentations conditions. Classical “batch fermentation” is a closed system, wherein the compositions of the medium is set at the beginning of the fermentation and is not subject to artificial alternations during the fermentation. A variation of the batch system is a “fed-batch fermentation” which also finds use in the present invention. In this variation, the substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression is likely to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the medium. Batch and fed-batch fermentations are common and well known in the art. “Continuous fermentation” is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth. Continuous fermentation systems strive to maintain steady state growth conditions. Methods for modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology.

In some embodiments of the present invention, cell-free transcription/translation systems find use in producing variant PGA(s). Several systems are commercially available and the methods are well-known to those skilled in the art.

The present invention provides methods of making variant PGA polypeptides or biologically active fragments thereof. In some embodiments, the method comprises: providing a host cell transformed with a polynucleotide encoding an amino acid sequence that comprises at least about 70% (or at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) sequence identity to SEQ ID NO: 12, 24, 40, 56, 70, 82, 100, 108, 110, 116, 136, 154, and/or 160, and comprising at least one mutation as provided herein; culturing the transformed host cell in a culture medium under conditions in which the host cell expresses the encoded variant PGA polypeptide; and optionally recovering or isolating the expressed variant PGA polypeptide, and/or recovering or isolating the culture medium containing the expressed variant PGA polypeptide. In some embodiments, the methods further provide optionally lysing the transformed host cells after expressing the encoded PGA polypeptide and optionally recovering and/or isolating the expressed variant PGA polypeptide from the cell lysate. The present invention further provides methods of making a variant PGA polypeptide comprising cultivating a host cell transformed with a variant PGA polypeptide under conditions suitable for the production of the variant PGA polypeptide and recovering the variant PGA polypeptide. Typically, recovery or isolation of the PGA polypeptide is from the host cell culture medium, the host cell or both, using protein recovery techniques that are well known in the art, including those described herein. In some embodiments, host cells are harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including, but not limited to freeze-thaw cycling, sonication, mechanical disruption, and/or use of cell lysing agents, as well as many other suitable methods well known to those skilled in the art.

Engineered PGA enzymes expressed in a host cell can be recovered from the cells and/or the culture medium using any one or more of the well-known techniques for protein purification, including, among others, lysozyme treatment, sonication, filtration, salting-out, ultra-centrifugation, and chromatography. Suitable solutions for lysing and the high efficiency extraction of proteins from bacteria, such as E. coli, are commercially available under the trade name CelLytic B™ (Sigma-Aldrich).

Thus, in some embodiments, the resulting polypeptide is recovered/isolated and optionally purified by any of a number of methods known in the art. For example, in some embodiments, the polypeptide is isolated from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, chromatography (e.g., ion exchange, affinity, hydrophobic interaction, chromatofocusing, and size exclusion), or precipitation. In some embodiments, protein refolding steps are used, as desired, in completing the configuration of the mature protein. In addition, in some embodiments, high performance liquid chromatography (HPLC) is employed in the final purification steps. For example, in some embodiments, methods known in the art, find use in the present invention (See e.g., Parry et al., Biochem. J., 353:117 [2001]; and Hong et al., Appl. Microbiol. Biotechnol., 73:1331 [2007], both of which are incorporated herein by reference). Indeed, any suitable purification methods known in the art find use in the present invention.

Chromatographic techniques for isolation of the PGA polypeptide include, but are not limited to reverse phase chromatography high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying a particular enzyme will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., are known to those skilled in the art.

In some embodiments, affinity techniques find use in isolating the improved PGA enzymes. For affinity chromatography purification, any antibody which specifically binds the PGA polypeptide may be used. For the production of antibodies, various host animals, including but not limited to rabbits, mice, rats, etc., may be immunized by injection with the PGA. The PGA polypeptide may be attached to a suitable carrier, such as BSA, by means of a side chain functional group or linkers attached to a side chain functional group. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacillus Calmette Guerin) and Corynebacterium parvum.

In some embodiments, the PGA variants are prepared and used in the form of cells expressing the enzymes, as crude extracts, or as isolated or purified preparations. In some embodiments, the PGA variants are prepared as lyophilisates, in powder form (e.g., acetone powders), or prepared as enzyme solutions. In some embodiments, the PGA variants are in the form of substantially pure preparations.

In some embodiments, the PGA polypeptides are attached to any suitable solid substrate. Solid substrates include but are not limited to a solid phase, surface, and/or membrane. Solid supports include, but are not limited to organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well as co-polymers and grafts thereof. A solid support can also be inorganic, such as glass, silica, controlled pore glass (CPG), reverse phase silica or metal, such as gold or platinum. The configuration of the substrate can be in the form of beads, spheres, particles, granules, a gel, a membrane or a surface. Surfaces can be planar, substantially planar, or non-planar. Solid supports can be porous or non-porous, and can have swelling or non-swelling characteristics. A solid support can be configured in the form of a well, depression, or other container, vessel, feature, or location. A plurality of supports can be configured on an array at various locations, addressable for robotic delivery of reagents, or by detection methods and/or instruments.

In some embodiments, immunological methods are used to purify PGA variants. In one approach, antibody raised against a variant PGA polypeptide (e.g., against a polypeptide comprising any of SEQ ID NOS: 2, 4, 12, 24, 40, 56, 70, 82, 100, 108, 110, 116, 136, 154, and/or 160, and/or an immunogenic fragment thereof) using conventional methods is immobilized on beads, mixed with cell culture media under conditions in which the variant PGA is bound, and precipitated. In a related approach, immunochromatography finds use.

In some embodiments, the variant PGAs are expressed as a fusion protein including a non-enzyme portion. In some embodiments, the variant PGA sequence is fused to a purification facilitating domain. As used herein, the term “purification facilitating domain” refers to a domain that mediates purification of the polypeptide to which it is fused. Suitable purification domains include, but are not limited to metal chelating peptides, histidine-tryptophan modules that allow purification on immobilized metals, a sequence which binds glutathione (e.g., GST), a hemagglutinin (HA) tag (corresponding to an epitope derived from the influenza hemagglutinin protein; See e.g., Wilson et al., Cell 37:767 [1984]), maltose binding protein sequences, the FLAG epitope utilized in the FLAGS extension/affinity purification system (e.g., the system available from Immunex Corp), and the like. One expression vector contemplated for use in the compositions and methods described herein provides for expression of a fusion protein comprising a polypeptide of the invention fused to a polyhistidine region separated by an enterokinase cleavage site. The histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography; See e.g., Porath et al., Prot. Exp. Purif., 3:263-281 [1992]) while the enterokinase cleavage site provides a means for separating the variant PGA polypeptide from the fusion protein. pGEX vectors (Promega) may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to ligand-agarose beads (e.g., glutathione-agarose in the case of GST-fusions) followed by elution in the presence of free ligand.

Experimental

Various features and embodiments of the disclosure are illustrated in the following representative examples, which are intended to be illustrative, and not limiting.

In the experimental disclosure below, the following abbreviations apply: ppm (parts per million); M (molar); mM (millimolar), uM and μM (micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg (milligrams); ug and μg (micrograms); L and 1 (liter); ml and mL (milliliter); cm (centimeters); mm (millimeters); um and μm (micrometers); sec. (seconds); min(s) (minute(s)); h(s) and hr(s) (hour(s)); U (units); MW (molecular weight); rpm (rotations per minute); ° C. (degrees Centigrade); RT (room temperature); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); aa (amino acid); TB (Terrific Broth; 12 g/L bacto-tryptone, 24 g/L yeast extract, 4 mL/L glycerol, 65 mM potassium phosphate, pH 7.0, 1 mM MgSO₄); CAM (chloramphenicol); PMBS (polymyxin B sulfate); IPTG (isopropyl thiogalactoside); TFA (trifluoroacetic acid); CHES (2-cyclohexylamino)ethanesulfonic acid; HPLC (high performance liquid chromatography); FIOPC (fold improvement over positive control); HTP (high throughput); LB (Luria broth); Codexis (Codexis, Inc., Redwood City, Calif.); Sigma-Aldrich (Sigma-Aldrich, St. Louis, Mo.); Millipore (Millipore, Corp., Billerica Mass.); Difco (Difco Laboratories, BD Diagnostic Systems, Detroit, Mich.); Daicel (Daicel, West Chester, Pa.); Genetix (Genetix USA, Inc., Beaverton, Oreg.); Molecular Devices (Molecular Devices, LLC, Sunnyvale, Calif.); Applied Biosystems (Applied Biosystems, part of Life Technologies, Corp., Grand Island, N.Y.), Agilent (Agilent Technologies, Inc., Santa Clara, Calif.); Thermo Scientific (part of Thermo Fisher Scientific, Waltham, Mass.); (Infors; Infors-HT, Bottmingen/Basel, Switzerland); Corning (Corning, Inc., Palo Alto, Calif.); and Bio-Rad (Bio-Rad Laboratories, Hercules, Calif.); Microfluidics (Microfluidics Corp., Newton, Mass.).

Example 1 E. coli Expression Hosts Containing Recombinant PGA Genes

The initial PGA enzymes used to produce the variants of the present invention were obtained from either the Codex® Acylase Panel (“PGA panel plate”; Codexis) or variants disclosed in co-owned US Pat. Appln. Publ. No. 2016/0326508. The PGA panel plate comprises a collection of engineered PGA polypeptides that have improved properties, as compared to the wild-type Kluyvera citrophila PGA. The wild type PGA gene is a heterodimer consisting of an alpha subunit (23.8 KDa) and a beta subunit (62.2KDa) that are linked by 54aa spacer region. Due to the presence of the spacer region, an autoprocessing step is required to form the active protein. During the development of the present invention, the wild-type gene was modified to eliminate the spacer region, thus eliminating the auto processing step. The PGA panel plate (Codexis) contains PGA variants that lack the spacer region (See e.g., US Pat. Appln. Publn. 2010/0143968 A1). The PGA-encoding genes were cloned into the expression vector pCK110900 (See, FIG. 3 of US Pat. Appln. Pubn. No. 2006/0195947), operatively linked to the lac promoter under control of the lacl repressor. The expression vector also contains the P15a origin of replication and a chloramphenicol resistance gene. The resulting plasmids were transformed into E. coli W3110, using standard methods known in the art. The transformants were isolated by subjecting the cells to chloramphenicol selection, as known in the art (See e.g., U.S. Pat. No. 8,383,346 and W2010/144103).

Example 2 Preparation of HTP PGA-Containing Wet Cell Pellets

E. coli cells containing recombinant PGA-encoding genes from monoclonal colonies were inoculated into 180p LB containing 1% glucose and 30 pg/mL chloramphenicol in the wells of 96 well shallow-well microtiter plates. The plates were sealed with O₂-permeable seals and cultures were grown overnight at 30° C., 200 rpm and 85% humidity. Then, 10₁ of each of the cell cultures were transferred into the wells of 96 well deep-well plates containing 390 mL TB and 30 pg/mL CAM. The deep-well plates were sealed with O₂-permeable seals and incubated at 30° C., 250 rpm and 85% humidity until OD₆₀₀ 0.6-0.8 was reached. The cell cultures were then induced by IPTG to a final concentration of 1 mM and incubated overnight under the same conditions as originally used. The cells were then pelleted using centrifugation at 4000 rpm for 10 min. The supernatants were discarded and the pellets frozen at −80° C. prior to lysis.

Example 3 Preparation of HTP PGA-Containing Cell Lysates

First, 200 μl lysis buffer containing 10 mM Tris-HCl buffer, pH 7.5, 1 mg/mL lysozyme, and 0.5 mg/mL PMBS was added to the cell paste in each well produced as described in Example 2. The cells were lysed at room temperature for 2 hours with shaking on a bench top shaker. The plate was then centrifuged for 15 min at 4000 rpm and 4° C. The clear supernatants were then used in biocatalytic reactions to determine their activity levels.

Example 4 Preparation of Lyophilized Lysates from Shake Flask (SF) Cultures

Selected HTP cultures grown as described above were plated onto LB agar plates with 1% glucose and 30 pg/ml CAM, and grown overnight at 37° C. A single colony from each culture was transferred to 6 ml of LB with 1% glucose and 30pg/ml CAM. The cultures were grown for 18 h at 30° C., 250 rpm, and subcultured approximately 1:50 into 250 ml of TB containing 30 pg/ml CAM, to a final OD₆₀₀ of 0.05. The cultures were grown for approximately 195 minutes at 30° C., 250 rpm, to an OD₆₀₀ between 0.6-0.8 and induced with 1 mM IPTG. The cultures were then grown for 20 h at 30° C., 250 rpm. The cultures were centrifuged 4000 rpm×20 min. The supernatant was discarded, and the pellets were resuspended in 30 ml of 20 mM TRIS-HCl, pH 7.5. The cells were pelleted (4000 rpm×20 min) and frozen at −80° C. for 120 minutes. Frozen pellets were resuspended in 30 ml of 20 mM TRIS-HCl pH 7.5, and lysed using a Microfluidizer® processor system (Microfluidics) at 18,000 psi. The lysates were pelleted (10,000 rpm×60 min) and the supernatants were frozen and lyophilized to generate shake flake (SF) enzymes.

Example 5 Improvements Over SEQ ID NO: 4 in the Acylation of Insulin at the A1, B1, and B29 Positions

SEQ ID NO: 4 was selected as the parent enzyme based on the results of screening variants disclosed in co-owned US Pat. Appln. Publ. No. 2016/0326508, for the production of the B29 deacylated product. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis, and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2 and the soluble lysate was generated as described in Example 3.

Each reaction well contained 200 μL of 0.1 M CHES, pH 10, 10 g/L insulin, 17 g/L methyl phenylacetate, and 20 μl HTP supernatant. The HTP plates were incubated in a Thermotron®shaker (3 mm throw, model # AJ185, Infors) at 30° C., 300 rpm, for 20 hours. The reactions were quenched with 200 μl acetonitrile and mixed for 5 minutes using a bench top shaker. The plates were then centrifuged at 4000 rpm for 5 minutes and loaded into an HPLC for analysis.

Activity relative to SEQ ID NO:4 (Activity FOP) was calculated as the percent conversion of the product formed by the variant over the percent conversion produced by SEQ ID NO: 4. The results are shown in Table 5.1. The percent conversion was calculated by dividing the area of the product peak by the sum of the areas of the substrate, product and impurities/side product peaks as observed by the HPLC analysis. Table 5.2 provides results showing the selectivity of the variants relative to SEQ ID NO:4.

TABLE 5.1 Activity of Variants Relative to SEQ ID NO: 4 Amino Acid Acylation Percent Conversion FIOP¹ at Variant SEQ ID Differences (Relative Designated Sites (Relative to SEQ ID NO: 4) NO: NO: (nt/aa) to SEQ ID NO: 4) A1 B29 B1 A1/B29 A1/B1 B1/B29 4 11/12 F71G; G74D +++ +++ ++++ ++++ ++++ ++++ 5 17/18 F24Y; V28A; F71C; F701W + ++++ ¹Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 4 and defined as follows: “+” > than 1.2-fold but less than 2.5-fold increased activity; “++” > than 2.5-fold but less than 5-fold increased activity; “+++” > than 5-fold increased activity but less than 10-fold; “++++” > than 10 fold.

TABLE 5.2 Selectivity of Variants Relative to SEQ ID NO: 4 Amino Acid Acylation Percent Selectivity (FIOP)¹ at Variant SEQ ID Differences (Relative Designated Sites (Relative to SEQ ID NO: 4) NO: NO: (nt/aa) to SEQ ID NO: 4) A1 B29 B1 A1/B29 A1/B1 B1/B29 3 85/86 F71R + 4 11/12 F71G; G74D + ++++ ++++ ++++ ++++ 5 17/18 F24Y; V28A; F71C; F701W ++++ ¹Levels of increased selectivity were determined relative to the reference polypeptide of SEQ ID NO: 4 and defined as follows: “+” > than 1.2-fold but less than 2.5-fold increased selectivity; “++” > than 2.5-fold but less than 5-fold increased selectivity; “+++” > than 5-fold increased selectivity but less than 10-fold; “++++” > than 10 fold.

Example 6 Improvement in the Acylation of Insulin at A1, B1 and B29 Positions Compared to SEQ ID NO: 12 in High Throughput Screening

SEQ ID NO: 12 was selected as the next parent enzyme, based on the results described in Example 5, (i.e., the best enzyme identified at acylating insulin at position B29). Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the soluble lysate was generated as described in Example 3.

Each variant was screened in a 200 μL reaction comprised of 10 g/L insulin, 0.1 M TRIS buffer pH9.25, 20% acetonitrile, 17 g/L methyl phenylacetate, and 10 μL clarified lysate, for 5 hours at 30° C. The 96-well plates were heat-sealed and incubated in a Thermotron® shaker at 100 rpm. The reactions were quenched with 200 μl acetonitrile and mixed for 5 minutes using a bench top shaker. The plates were then centrifuged at 4000 rpm for 5 minutes and loaded into an HPLC for analysis.

The percent conversion relative to SEQ ID NO:12 (Percent Conversion FIOP) was calculated as the percent conversion of the product formed by the variant over the percent conversion produced by SEQ ID NO: 12. These results are shown in Tables 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, and 6.7. The percent conversion was calculated by dividing the area of the product peak by the sum of the areas of the substrate, product and impurities/side product peaks as observed by the HPLC analysis.

The percent selectivity relative to SEQ ID NO:12 (Percent Selectivity FIOP) was calculated as the percent selectivity of the product formed by the variant over the percent selectivity produced by SEQ ID NO: 12. The results are shown in Tables 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, and 6.7. The percent selectivity was calculated by dividing the area of the product peak by the sum of the areas of the product and impurities/side product peaks as observed by the HPLC analysis.

TABLE 6.1 Activity and Selectivity of Variants Acylating at the A1 Site Relative to SEQ ID NO: 12 Acylation Percent Acylation Percent Conversion (FIOP)¹ Selectivity (FIOP)¹ Variant SEQ ID Amino Acid Differences (Relative to at the A1 Site (Relative for the A1 Site (Relative NO: NO: (nt/aa) SEQ ID NO: 12) to SEQ ID NO: 12) to SEQ ID NO: 12) 6 Y27T; A255G; W370I; +++ 7 D623N; + 8 T384R; ++ + 9 L253S; + + 10 T705S; + 11 A373Y; + + 12 Y27T; F254W; A470V; ++ 13 Y27T; L253V; A255G; N348R; + 14 Y27T; D74S; F254W; A255G; N348R; K369C; + + T384P; 15 Y27T; D74N; L253V; F254W; N348R; K369C; ++ + T384P; 16 L253M; + + 17 N457T; + + 18 R317S; Q380P; + + 19 69/70 K128W; + + ¹Levels of increased activity or selectivity were determined relative to the reference polypeptide of SEQ ID NO: 12 and defined as follows: “+” > than 1.0-fold but less than 1.5-fold increase; “++” > than 1.5-fold but less than 2.0-fold; “+++” > than 2.0-fold.

TABLE 6.2 Activity and Selectivity of Variants Acylating at the B29 Site Relative to SEQ ID NO: 12 Acylation Percent Acylation Percent Conversion (FIOP)¹ Selectivity (FIOP)² Variant SEQ ID Amino Acid Differences (Relative to at the B29 Site (Relative for the B29 Site (Relative NO: NO: (nt/aa) SEQ ID NO: 12) to SEQ ID NO: 12) to SEQ ID NO: 12) 20 Y27T; N348R; D381K; + + 21 Y27T; D74S; A255G; N348R; K369C; D381K; + 22 15/16 Y27T; D74S; A255G; N348R; D381K; T384P; + + 23 Y27T; N348R; K369C; W370I; D381K; T384P; + 24 Y27T; D74G; F254W; A255G; N348R; K369C; + W370I; D381K; 25 Y27T; F254W; A255G; N348R; W370I; D381K; + 26 13/14 Y27T; A255G; N348R; W370I; D381K; T384P; + 27 D381F; + + 28 Q134M; + + 29 D623W; ++ + 30 E253R; + + 31 N627M; + + 32 N627R; + + 33 D623N; ++ + 34 K615V; + + 35 D381E; + + 36 D381R; + ++ 37 A132G; + + 38 A467S; + + 39 F256Y; + + 40 D623V; + + 41 K615H; + 42 D623A; + + 43 D381Q; + + 44 K615C; + 45 T384R; + + 46 F256H; + + 47 T453C; + 48 D381V; + 49 D381K; + + 50 D381F; Q672K; + 51 D623Y; + + 53 D623R; +++ ++ 54 D623F; + + 55 D623K; +++ ++ 56 D381I; + 57 A373K; +++ ++ 58 S706K; + ++ 59 N348K; A467T; + + 60 D709G; + + 61 D709A; + + 62 F620R; + + 63 D709N; + ++ 64 D709H; + + 65 E377A; + 66 F620K; + + 67 S706R; + 68 21/22 D709R; + ++++ 69 N20S; D709Q; + 70 D709S; + + 71 V618C; + + 72 A69M; + 73 F254K; + + 74 A84V; + 75 F701H; + 76 P383K; + 77 A69E; ++ 78 I708V; + 79 A255K; ++ ++ 80 A255R; ++ ++ 81 A69V; + 82 P383R; ++++ ¹Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 12 and defined as follows: “+” > than 2.0-fold but less than 4.0-fold increase; “++” > than 4.0-fold but less than 6.0-fold; “+++” > than 6.0-fold. ²Levels of increased selectivity were determined relative to the reference polypeptide of SEQ ID NO: 12 and defined as follows: “+” > than 2.0-fold but less than 5.0-fold increase; “++” > than 5.0-fold but less than 10.0-fold; “+++” > than 10.0-fold but less than 15-fold; “++++“ > than 15-fold.

TABLE 6.3 Activity and Selectivity of Variants Acylating at the A1 and B29 Sites Relative to SEQ ID NO: 12 Acylation Percent Acylation Percent Conversion (FIOP)¹ Selectivity (FIOP)² at the A1 and B29 for the A1 and B29 Variant SEQ ID Amino Acid Differences (Relative to Sites (Relative to Sites (Relative to NO: NO: (nt/aa) SEQ ID NO: 12) SEQ ID NO: 12) SEQ ID NO: 12) 198 Y27T; D74S; F254W; N348R; D381W; + + 199 Y27T; A255G; N348R; W370I; + 200 Y27T; N348R; T384P; + + 201 Y27T; D74G; F254W; A255G; N348R; D381W; + + 202 Y27T; D74S; N348R; + 203 Y27T; A255G; N348R; + + 204 Y27T; L253V; N348R; D381F; T384P; + + 205 Y27T; L253V; F254W; N348R; T384P; + 206 Y27T; D74P; F254W; A255G; N348R; D381K; + + T384P; 207 Y27T; N348R; + + 208 Y27T; F254W; A255G; N348R; W370I; D381W; + T384P; 22 15/16 Y27T; D74S; A255G; N348R; D381K; T384P; + + 209 Y27T; D74G; F254W; N348R; D381W; T384P; + + 210 Y27T; F254W; A255G; D381K; T384P; + + 211 Y27T; D74S; F254W; N348R; D381F; + + 212 Y27T; A255G; N348R; D381W; T384P; + + 213 Y27T; F254W; N348R; D381W; T384P; + + 214 Y27T; D74G; N348R; + + 181 Y27T; F254W; A255G; T384P; ++ + 182 Y27T; F254W; A255G; ++ + 215 Y27T; D74S; L253V; N348R; +++ ++ 183 Y27T; L253V; F254W; A255G; N348R; D381F; + + T384P; 184 Y27T; F254W; A255G; N348R; + + 185 Y27T; D74N; F254W; T384P; +++ + 186 Y27T; L253V; N348R; + 187 Y27T; D74G; A255G; N348R; ++ ++ 12 Y27T; F254W; A470V; + 13 Y27T; L253V; A255G; N348R; + + 189 Y27T; D74G; L253V; A255G; N348R; D381F; ++ + 6 Y27T; A255G; W370I; ++ + 190 Y27T; D74S; F254W; A255G; N348R; +++ ++ 216 Y27T; D74N; L253V; F254W; + 191  9/10 Y27T; D74S; L253V; F254W; N348R; D381W; ++ ++ T384P; 192 Y27T; D74N; F254W; A255G; N348R; +++ ++ 217 5/6 Y27T; D74G; L253V; F254W; A255G; N348R; +++ + 194 Y27T; L253V; N348R; W370I; D381F; T384P; + + 26 13/14 Y27T; A255G; N348R; W370I; D381K; T384P; + + 195 Y27T; D74S; A255G; W370I; ++ + 29 D623W; + + 30 L253R; + + 42 D623A; + + 218 D623N; + + 46 F256H; + + 219 A616R; + 220 D623L; + + 51 D623Y; + + 54 D623F; + + 55 D623K; + + 221 D381Q; + + 222 T384R; ++ ++ 223 19/20 D623R; ++ +++ 57 A373K; + + 224 H472R; + 225 F620R; + + 197 A255P; + 73 F254K; + + 80 A255R; + + ¹Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 12 and defined as follows: “+” > than 2.0-fold but less than 5.0-fold increase; “++” > than 5.0-fold but less than 10.0-fold; “+++” > than 10.0-fold. ²Levels of increased selectivity were determined relative to the reference polypeptide of SEQ ID NO: 12 and defined as follows: “+” > than 2.0-fold but less than 5.0-fold increase; “++” > than 5.0-fold but less than 7.0-fold; “+++” > than 7.0-fold.

TABLE 6.4 Activity and Selectivity of Variants Acylating at the A1 and B1 Sites Relative to SEQ ID NO: 12 Acylation Percent Acylation Percent Conversion (FIOP)¹ Selectivity (FIOP)² at the A1 and B1 for the A1 and B1 Variant SEQ ID Amino Acid Differences (Relative to Sites (Relative to Sites (Relative to NO: NO: (nt/aa) SEQ ID NO: 12) SEQ ID NO: 12) SEQ ID NO: 12) 226 Y27T; L253V; A255G; N348R; T384P; + + 83 Y27T; L253V; + + 84 Y27T; D74G; L253V; F254W; + + 85 Y27T; D74G; L253V; A255G; N348R; W370I; + + T384P; 86 Y27T; D74N; L253V; F254W; N348R; W370I; + + D381K; T384P; 87 Y27T; D74N; L253V; F254W; A255G; N348R; + + W370I; 199 Y27T; A255G; N348R; W370I; + + 227 Y27T; D74P; L253V; F254W; A255G; N348R; + + K369C; W370I; 88 Y27T; D74G; L253V; N348R; K369C; W370I; + + D381F; T384P; 89 Y27T; L253V; F254W; N348R; + + 90 Y27T; D74P; L253V; F254W; A255G; N348R; + + 228 Y27T; + 229 Y27T; D74G; L253V; N348R; K369C; W370I; + + 91 Y27T; D74G; L253V; F254W; A255G; W370I; ++ + 92 Y27T; L253V; F254W; A255G; + + 93 Y27T; D74G; L253V; F254W; T384P; + + 94 Y27T; D74N; F254W; N348R; W370I; + + 95 Y27T; D74S; L253V; N348R; W370I; D381K; + + T384P; 96 Y27T; L253V; A255G; W370I; + + 97 Y27T; D74G; F254W; A255G; D381F; + + 98 Y27T; L253V; K369C; W370I; + + 230 Y27T; D74P; F254W; A255G; N348R; + + 99 Y27T; L253V; F254W; N348R; D381F; + + 101 Y27T; D74S; L253V; A255G; T384P; + + 231 Y27T; D74G; A255G; W370I; + + 232 Y27T; D74S; N348R; + 102 Y27T; D74N; L253V; N348R; W370I; D381W; + + T384P; 233 Y27T; D74G; L253V; F254W; N348R; + + 103 Y27T; D74P; L253V; N348R; + + 234 Y27T; L253V; F254W; A255G; N348R; + + 235 Y27T; D74S; L253V; F254W; A255G; N348R; + + 104 Y27T; D74G; F254W; A255G; N348R; K369C; + + W370I; D381F; 105 Y27T; D74P; F254W; A255G; N348R; K369C; + + W370I; 106 65/66 Y27T; D74N; L253V; F254W; W370I; D381K; ++ + 236 Y27T; D74G; F254W; K369C; W370I; + + 237 Y27T; D74G; L253V; N348R; W370I; + + 107 Y27T; D74G; L253V; T384P; + + 108 Y27T; D74S; N348R; W370I; + + 109 Y27T; D74S; L253V; F254W; A255G; K369C; + + W370I; 238 Y27T; D74G; F254W; A255G; N348R; + + 239 Y27T; L253V; N348R; W370I; T384P; + + 111 Y27T; D74N; L253V; F254W; A255G; + + 240 Y27T; L253V; F254W; + + 241 Y27T; D74G; K369C; W370I; + + 242 Y27T; F254W; A449V; + + 114 Y27T; D74G; L253V; A255G; N348R; D381W; + + 115 Y27T; D74P; L253V; W370I; + + 117 Y27T; D74G; L253V; F254W; A255G; W370I; ++ + D381K; T384P; 118 Y27T; D74N; F254W; A255G; N348R; W370I; + + D381K; 119 Y27T; D74S; L253V; N348R; K369C; W370I; + + T384P; 243 Y27T; D74N; L253V; N348R; + + 120 Y27T; D74N; L253V; F254W; N348R; W370I; + + D381F; 121 Y27T; D74S; F254W; A255G; N348R; W370I; + + D381F; T384P; 122 Y27T; D74G; L253V; F254W; A255G; D381K; + + 123 Y27T; D74S; F254W; K369L; W370I; + + 124 Y27T; L253V; F254W; D381F; T384P; + + 125 Y27T; D74G; L253V; F254W; A255G; K369C; + + W370I; 126 Y27T; D74G; F254W; A255G; N348R; W370I; + + 127 Y27T; D74G; L253V; F254W; A255G; N348R; + + W370I; T384P; 128 L253V; N348R; W370I; + + 244 Y27T; F254W; N348R; W370I; + + 129 Y27T; D74N; L253V; F254W; K369C; + + 131 Y27T; D74S; L253V; N348R; D381W; + + 245 Y27T; D74S; A255G; + + 134 Y27T; D74S; L253V; A255G; N348R; W370I; + + D381K; 136 Y27T; D74N; L253V; F254W; A255G; N348R; + + W370I; T384P; 137 Y27T; D74S; F254W; A255G; W370I; ++ + 138 Y27T; F254W; A255G; K369C; W370I; D381F; + + T384P; 139 Y27T; L253V; F254W; D381F; + + 140 Y27T; D74N; F254W; N348R; + + 141 67/68 Y27T; F254W; A255G; W370I; ++ + 142 Y27T; D74N; A107V; A255G; N348R; K369C; + + W370I; 143 Y27T; F254W; A255G; N348R; W370I; + + 144 Y27T; D74N; F254W; + + 246 Y27T; D74S; F254W; K369C; T384P; + + 145 Y27T; D74G; L253V; D381F; T384P; + + 146 Y27T; D74P; L253V; A255G; + + 247 Y27T; D74G; A255G; N348R; K369C; W370I; + + 148 Y27T; D74S; F254W; A255G; N348R; K369C; + + W370I; 248 Y27T; F254W; A255G; N348R; K369C; W370I; + + 149 Y27T; D74P; W370I; + + 249 Y27T; D74P; L253V; F254W; N348R; K369C; + + W370I; 150 Y27T; L253V; F254W; T384P; + + 205 Y27T; L253V; F254W; N348R; T384P; + + 151 Y27T; D74N; L253V; F254W; A255G; W370I; + + 250 Y27T; F254W; A255G; N348R; + + 251 Y27T; D74S; F254W; N348R; + + 153 Y27T; F254W; A255G; N348R; W370I; T384P; + + 252 D74N; L253V; F254W; K369C; W370I; + + 253 Y27T; D74S; K369C; W370I; D381K; T384P; + + 155 Y27T; L253V; A255G; W370I; D381F; T384P; ++ + 157 Y27T; L253V; F254W; N348R; W370I; T384P; + + 158 Y27T; D74N; F254W; N348R; W370I; D381K; + + 159 Y27T; D74N; L253V; A255G; N348R; K369C; + + W370I; 160 Y27T; L253V; F254W; N348R; D381W; T384P; + + 161 Y27T; L253V; F254W; A255G; G260C; N348R; + D381F; T384P; 162 Y27T; D74P; L253V; F254W; N348R; D381F; + + T384P; 165 Y27T; D74G; L253V; A255G; N348R; T384P; + + 166 Y27T; D74N; L253V; A255G; W370I; + + 167 Y27T; D74N; A255G; N348R; W370I; + + 208 Y27T; F254W; A255G; N348R; W370I; D381W; + + T384P; 254 Y27T; L253V; A255G; N348R; K369C; W370I; + + 169 Y27T; L253V; D381F; T384P; + + 170 Y27T; D74P; L253V; F254W; N348R; W370I; + + D381W; T384P; 171 Y27T; L253V; F254W; A255G; N348R; W370I; + + T384P; 255 A255G; N348R; W370I; + + 209 Y27T; D74G; F254W; N348R; D381W; T384P; + 210 Y27T; F254W; A255G; D381K; T384P; + + 256 Y27T; D74N; F254W; A255G; K369C; + + 257 Y27T; D74P; L253V; F254W; N348R; + + 173 Y27T; L253V; F254W; N348R; W370I; D38IF; + + 174 Y27T; D74G; L253V; A255G; + + 175 Y27T; D74N; L253V; F254W; N348R; W370I; + + 177 Y27T; D74G; L253V; F254W; A255G; N348R; + + K369C; W370I; D381F; 178 Y27T; D74G; L253V; F254W; N348R; K369C; + + W370I; 258 Y27T; L253V; F254W; A255G; N348R; W370I; ++ + 179 Y27T; D74N; L253V; + + 181 Y27T; F254W; A255G; T384P; ++ ++ 182 Y27T; F254W; A255G; ++ ++ 215 Y27T; D74S; L253V; N348R; ++ ++ 183 Y27T; L253V; F254W; A255G; N348R; D381F; + + T384P; 185 Y27T; D74N; F254W; T384P; +++ ++ 186 Y27T; L253V; N348R; + + 187 Y27T; D74G; A255G; N348R; + + 12 Y27T; F254W; A470V; + + 13 Y27T; L253V; A255G; N348R; + + 189 Y27T; D74G; L253V; A255G; N348R; D381F; + + 6 Y27T; A255G; W370I; ++ ++ 190 Y27T; D74S; F254W; A255G; N348R; ++ + 216 Y27T; D74N; L253V; F254W; ++++ ++ 191  9/10 Y27T; D74S; L253V; F254W; N348R; D381W; ++ ++ T384P; 192 Y27T; D74N; F254W; A255G; N348R; ++ + 193 Y27T; D74P; L253V; F254W; A255G; +++ ++ 217 5/6 Y27T; D74G; L253V; F254W; A255G; N348R; ++ ++ 14 Y27T; D74S; F254W; A255G; N348R; K369C; + T384P; 15 Y27T; D74N; L253V; F254W; N348R; K369C; + + T384P; 194 Y27T; L253V; N348R; W370I; D381F; T384P; + + 195 Y27T; D74S; A255G; W370I; +++ ++ 27 D381F; + + 259 A132S; + + 28 Q134M; + + 29 D623W; + + 260 T131L; + + 35 D381L; + + 37 A132G; + + 262 W370V; + + 263 D381R; + + 264 T384R; + + 265 D623Y; + + 7 D623N; + + 266 D623R; + + 267 S619I; + 268 L253V; + + 269 T133K; + 197 A255P; + + 270 I708M; + 271 F254T; + + 272 T705S; + ¹Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 12 and defined as follows: “+” > than 2.0-fold but less than 10.0-fold increase; “++” > than 10.0-fold but less than 50.0-fold; “+++” > than 50.0-fold but less than 100-fold; “++++” > than 100. ²Levels of increased selectivity were determined relative to the reference polypeptide of SEQ ID NO: 12 and defined as follows: “+” > than 2.0-fold but less than 10.0-fold increase; “++” > than 10.0-fold but less than 50.0-fold; “+++” > than 50.0-fold but less than 100-fold; “++++” > than 100.

TABLE 6.5 Activity and Selectivity of Variants Acylating at the A1 and B29 Sites Relative to SEQ ID NO: 12 Acylation Percent Acylation Percent Conversion (FIOP)¹ Selectivity (FIOP)² at the A1 and B29 for the A1 and B29 Variant SEQ ID Amino Acid Differences (Relative to Sites (Relative to Sites (Relative to NO: NO: (nt/aa) SEQ ID NO: 12) SEQ ID NO: 12) SEQ ID NO: 12) 85 Y27T; D74G; L253V; A255G; N348R; W370I; + + T384P; 86 Y27T; D74N; L253V; F254W; N348R; W370I; + + D381K; T384P; 87 Y27T; D74N; L253V; F254W; A255G; N348R; + + W370I; 91 Y27T; D74G; L253V; F254W; A255G; W370I; + 93 Y27T; D74G; L253V; F254W; T384P; + 95 Y27T; D74S; L253V; N348R; W370I; D381K; + + T384P; 102 Y27T; D74N; L253V; N348R; W370I; D381W; ++ + T384P; 233 Y27T; D74G; L253V; F254W; N348R; + 273 Y27T; L253V; F254W; A255G; N348R; K369C; + + W370I; D381W; T384P; 235 Y27T; D74S; L253V; F254W; A255G; N348R; + + 106 65/66 Y27T; D74N; L253V; F254W; W370I; D381K; + + 108 Y27T; D74S; N348R; W370I; + + 111 Y27T; D74N; L253V; F254W; A255G; + 113 Y27T; D74G; A255G; W370I; + + 114 Y27T; D74G; L253V; A255G; N348R; D381W; + + 115 Y27T; D74P; L253V; W370I; + + 117 Y27T; D74G; L253V; F254W; A255G; W370I; ++ + D381K; T384P; 274 Y27T; L253V; F254W; A255G; N348R; K369C; + + T384P; 118 Y27T; D74N; F254W; A255G; N348R; W370I; + + D381K; 120 Y27T; D74N; L253V; F254W; N348R; W370I; + + D381F; 121 Y27T; D74S; F254W; A255G; N348R; W370I; + + D381F; T384P; 122 Y27T; D74G; L253V; F254W; A255G; D381K; + + 126 Y27T; D74G; F254W; A255G; N348R; W370I; + + 127 Y27T; D74G; L253V; F254W; A255G; N348R; + + W370I; T384P; 131 Y27T; D74S; L253V; N348R; D381W; + + 245 Y27T; D74S; A255G; + 134 Y27T; D74S; L253V; A255G; N348R; W370I; + + D381K; 136 Y27T; D74N; L253V; F254W; A255G; N348R; + + W370I; T384P; 137 Y27T; D74S; F254W; A255G; W370I; + + 140 Y27T; D74N; F254W; N348R; + 141 67/68 Y27T; F254W; A255G; W370I; + 143 Y27T; F254W; A255G; N348R; W370I; + 145 Y27T; D74G; L253V; D381F; T384P; + + 149 Y27T; D74P; W370I; + 151 Y27T; D74N; L253V; F254W; A255G; W370I; + + 152 Y27T; D74G; L253V; N348R; W370I; + + 157 Y27T; L253V; F254W; N348R; W370I; T384P; + + 160 Y27T; L253V; F254W; N348R; D381W; T384P; + + 161 Y27T; L253V; F254W; A255G; G260C; N348R; + + D381F; T384P; 162 Y27T; D74P; L253V; F254W; N348R; D381F; + + T384P; 275 Y27T; D74G; L253V; F254W; + + 164 Y27T; D74N; L253V; N348R; + + 165 Y27T; D74G; L253V; A255G; N348R; T384P; + + 166 Y27T; D74N; L253V; A255G; W370I; + + 167 Y27T; D74N; A255G; N348R; W370I; + + 276 Y27T; D74P; F254W; A255G; N348R; D381K; + + T384P; 208 Y27T; F254W; A255G; N348R; W370I; D381W; + + T384P; 254 Y27T; L253V; A255G; N348R; K369C; W3701; + 22 15/16 Y27T; D74S; A255G; N348R; D381K; T384P; + + 170 Y27T; D74P; L253V; F254W; N348R; W370I; + + D381W; T384P; 171 Y27T; L253V; F254W; A255G; N348R; W370I; + + T384P; 209 Y27T; D74G; F254W; N348R; D381W; T384P; + + 277 Y27T; D74N; F254W; N348R; W370I; + + 278 Y27T; D74P; L253V; F254W; N348R; K369C; + + 257 Y27T; D74P; L253V; F254W; N348R; + 279 Y27T; F254W; K369C; D381F; T384P; + + 173 Y27T; L253V; F254W; N348R; W370I; D381F; + + 175 Y27T; D74N; L253V; F254W; N348R; W370I; + + 280 Y27T; F254W; N348R; K369C; W370I; D381N; + + T384P; 180 Y27T; D74G; L253V; F254W; A255G; N348R; + 281 D623N; + + 264 T384R; + + 220 D623L; + + 62 F620R; ++ +++ 63 D709N; +++ +++ 19 69/70 K128W; ++ ++ 282 T705E; ++ ++ 283 A255E; ++ ++ 284 F254T; ++ ++ ¹Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 12 and defined as follows: “+” > than 2.0-fold but less than 10.0-fold increase; “++” > than 10.0-fold but less than 50.0-fold; “+++” > than 50.0-fold but less than 100-fold; “++++” > than 100. ²Levels of increased selectivity were determined relative to the reference polypeptide of SEQ ID NO: 12 and defined as follows: “+” > than 2.0-fold but less than 10.0-fold increase; “++” > than 10.0-fold but less than 50.0-fold; “+++” > than 50.0-fold but less than 100-fold; “++++” > than 100.

TABLE 6.6 Activity and Selectivity of Variants Acylating at the A1, B1, and B29 Sites Relative to SEQ ID NO: 12 Acylation Percent Acylation Percent Conversion (FIOP)¹ Selectivity (FIOP)² at the A1, B1, and for the A1, B1, and Variant SEQ ID Amino Acid Differences (Relative to B29 Sites (Relative B29 Sites (Relative NO: NO: (nt/aa) SEQ ID NO: 12) to SEQ ID NO: 12) to SEQ ID NO: 12) 226 Y27T; L253V; A255G; N348R; T384P; + + 83 Y27T; L253V; + + 84 Y27T; D74G; L253V; F254W; + + 198 Y27T; D74S; F254W; N348R; D381W; + + 285 Y27T; D74G; A255G; N348R; K369C; D381F; + + T384P; 85 Y27T; D74G; L253V; A255G; N348R; W370I; + + T384P; 86 Y27T; D74N; L253V; F254W; N348R; W370I; ++ ++ D381K; T384P; 87 Y27T; D74N; L253V; F254W; A255G; N348R; + + W370I; 199 Y27T; A255G; N348R; W370I; ++ + 200 Y27T; N348R; T384P; + + 88 Y27T; D74G; L253V; N348R; K369C; W370I; + + D381F; T384P; 90 Y27T; D74P; L253V; F254W; A255G; N348R; + + 228 Y27T; + + 91 Y27T; D74G; L253V; F254W; A255G; W370I; + + 92 Y27T; L253V; F254W; A255G; + + 93 Y27T; D74G; L253V; F254W; T384P; + + 94 Y27T; D74N; F254W; N348R; W370I; + + 95 Y27T; D74S; L253V; N348R; W370I; D381K; ++ ++ T384P; 286 Y27T; L253V; N348R; + + 96 Y27T; L253V; A255G; W370I; + + 97 Y27T; D74G; F254W; A255G; D381F; + + 287 Y27T; F254W; N348R; T384P; + 201 Y27T; D74G; F254W; A255G; N348R; D381W; + + 288 D74N; F254W; A255G; N348R; T384P; + + 289 Y27T; F254W; A255G; + + 202 Y27T; D74S; N348R; + + 230 Y27T; D74P; F254W; A255G; N348R; + + 99 Y27T; L253V; F254W; N348R; D381F; + + 101 Y27T; D74S; L253V; A255G; T384P; + + 231 Y27T; D74G; A255G; W370I; ++ + 102 Y27T; D74N; L253V; N348R; W370I; D381W; ++ ++ T384P; 233 Y27T; D74G; L253V; F254W; N348R; ++ + 103 Y27T; D74P; L253V; N348R; + + 234 Y27T; L253V; F254W; A255G; N348R; + + 235 Y27T; D74S; L253V; F254W; A255G; N348R; + + 104 Y27T; D74G; F254W; A255G; N348R; K369C; + + W370I; D381F; 105 Y27T; D74P; F254W; A255G; N348R; K369C; + + W370I; 106 65/66 Y27T; D74N; L253V; F254W; W370I; D381K; ++ ++ 236 Y27T; D74G; F254W; K369C; W370I; + + 237 Y27T; D74G; L253V; N348R; W370I; ++ + 107 Y27T; D74G; L253V; T384P; + + 108 Y27T; D74S; N348R; W370I; ++ + 290 Y27T; D74G; A255G; N348R; + + 238 Y27T; D74G; F254W; A255G; N348R; + + 239 Y27T; L253V; N348R; W370I; T384P; ++ + 111 Y27T; D74N; L253V; F254W; A255G; + + 242 Y27T; F254W; A449V; + + 114 Y27T; D74G; L253V; A255G; N348R; D381W; + + 115 Y27T; D74P; L253V; W370I; + + 117 Y27T; D74G; L253V; F254W; A255G; W370I; ++ ++ D381K; T384P; 118 Y27T; D74N; F254W; A255G; N348R; W370I; + + D381K; 120 Y27T; D74N; L253V; F254W; N348R; W370I; ++ ++ D381F; 121 Y27T; D74S; F254W; A255G; N348R; W370I; ++ ++ D381F; T384P; 122 Y27T; D74G; L253V; F254W; A255G; D381K; ++ + 123 Y27T; D74S; F254W; K369L; W370I; + + 291 Y27T; D74N; F254W; A255G; N348R; K369C; + + D381F; 124 Y27T; L253V; F254W; D381F; T384P; ++ + 126 Y27T; D74G; F254W; A255G; N348R; W370I; ++ ++ 127 Y27T; D74G; L253V; F254W; A255G; N348R; + + W370I; T384P; 128 L253V; N348R; W370I; + + 244 Y27T; F254W; N348R; W370I; + + 131 Y27T; D74S; L253V; N348R; D381W; ++ ++ 132 Y27T; D74S; L253V; N348R; + + 245 Y27T; D74S; A255G; + + 292 Y27T; L253V; A255G; N348R; + + 134 Y27T; D74S; L253V; A255G; N348R; W370I; + + D381K; 136 Y27T; D74N; L253V; F254W; A255G; N348R; + + W370I; T384P; 137 Y27T; D74S; F254W; A255G; W370I; ++ + 138 Y27T; F254W; A255G; K369C; W370I; D381F; + + T384P; 203 Y27T; A255G; N348R; + + 139 Y27T; L253V; F254W; D381F; + + 140 Y27T; D74N; F254W; N348R; + + 141 67/68 Y27T; F254W; A255G; W370I; ++ ++ 204 Y27T; L253V; N348R; D381F; T384P; + + 143 Y27T; F254W; A255G; N348R; W370I; ++ ++ 293 Y27T; L253V; F254W; N348R; + + 144 Y27T; D74N; F254W; + + 294 Y27T; D74N; F254W; N348R; K369C; D381F; + + T384P; 145 Y27T; D74G; L253V; D381F; T384P; ++ ++ 146 Y27T; D74P; L253V; A255G; + + 148 Y27T; D74S; F254W; A255G; N348R; K369C; + + W370I; 149 Y27T; D74P; W370I; ++ + 150 Y27T; L253V; F254W; T384P; + + 295 Y27T; D74N; N348R; + + 205 Y27T; L253V; F254W; N348R; T384P; + + 151 Y27T; D74N; L253V; F254W; A255G; W370I; + + 250 Y27T; F254W; A255G; N348R; + + 251 Y27T; D74S; F254W; N348R; + + 206 Y27T; D74P; F254W; A255G; N348R; D381K; + + T384P; 153 Y27T; F254W; A255G; N348R; W370I; T384P; ++ ++ 296 7/8 Y27T; D74N; N348R; T384P; + + 253 Y27T; D74S; K369C; W370I; D381K; T384P; + + 155 Y27T; L253V; A255G; W370I; D381F; T384P; ++ ++ 157 Y27T; L253V; F254W; N348R; W370I; T384P; ++ + 158 Y27T; D74N; F254W; N348R; W370I; D381K; + + 160 Y27T; L253V; F254W; N348R; D381W; T384P; ++ + 297 Y27T; L253V; F254W; + + 161 Y27T; L253V; F254W; A255G; G260C; N348R; + + D381F; T384P; 162 Y27T; D74P; L253V; F254W; N348R; D381F; ++ ++ T384P; 164 Y27T; D74N; L253V; N348R; + + 165 Y27T; D74G; L253V; A255G; N348R; T384P; + + 166 Y27T; D74N; L253V; A255G; W370I; + + 167 Y27T; D74N; A255G; N348R; W370I; + + 207 Y27T; N348R; + + 208 Y27T; F254W; A255G; N348R; W370I; D381W; ++ ++ T384P; 169 Y27T; L253V; D381F; T384P; + + 22 15/16 Y27T; D74S; A255G; N348R; D381K; T384P; + + 170 Y27T; D74P; L253V; F254W; N348R; W370I; ++ ++ D381W; T384P; 171 Y27T; L253V; F254W; A255G; N348R; W370I; ++ + T384P; 255 A255G; N348R; W370I; + + 209 Y27T; D74G; F254W; N348R; D381W; T384P; ++ ++ 210 Y27T; F254W; A255G; D381K; T384P; ++ ++ 211 Y27T; D74S; F254W; N348R; D381F; + + 257 Y27T; D74P; L253V; F254W; N348R; + + 173 Y27T; L253V; F254W; N348R; W370I; D381F; + + 174 Y27T; D74G; L253V; A255G; + + 175 Y27T; D74N; L253V; F254W; N348R; W370I; + + 177 Y27T; D74G; L253V; F254W; A255G; N348R; + + K369C; W370I; D381F; 178 Y27T; D74G; L253V; F254W; N348R; K369C; + + W370I; 212 Y27T; A255G; N348R; D381W; T384P; + + 258 Y27T; L253V; F254W; A255G; N348R; W370I; ++ + 213 Y27T; F254W; N348R; D381W; T384P; + + 214 Y27T; D74G; N348R; + + 179 Y27T; D74N; L253V; + + 180 Y27T; D74G; L253V; F254W; A255G; N348R; + + 29 D623W; + + 40 D623V; + + 42 D623A; + + 43 D381Q; + + 220 D623L; + + 298 D623N; + + 8 T384R; ++ + 57 A373K; + + 299 F620R; + + 300 F254T; + + 197 A255P; + + ¹Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 12 and defined as follows: “+” > than 2.0-fold but less than 10.0-fold increase; “++” > than 10.0-fold but less than 50.0-fold; “+++” > than 50.0-fold but less than 100-fold; “++++” > than 100. ²Levels of increased selectivity were determined relative to the reference polypeptide of SEQ ID NO: 12 and defined as follows: “+” > than 2.0-fold but less than 10.0-fold increase; “++” > than 10.0-fold but less than 50.0-fold; “+++” > than 50.0-fold but less than 100-fold; “++++” > than 100.

TABLE 6.7 Activity and Selectivity of Variants Acylating at the B1 Site Relative to SEQ ID NO: 12 Acylation Acylation Percent Percent Conversion Selectivity (FIOP)¹ at the (FIOP)² for the SEQ ID B1 Site B1 Site Variant NO: Amino Acid Differences (Relative to (Relative to NO: (nt/aa) (Relative to SEQ ID NO: 12) SEQ ID NO: 12) SEQ ID NO: 12) 83 Y27T; L253V; + 84 Y27T; D74G; L253V; F254W; ++ + 85 Y27T; D74G; L253V; A255G; N348R; W370I; T384P; ++ + 86 Y27T; D74N; L253V; F254W; N348R; W370I; ++ ++ D381K; T384P; 87 Y27T; D74N; L253V; F254W; A255G; N348R; W3701; ++ ++ 88 Y27T; D74G; L253V; N348R; K369C; W370I; + + D381F; T384P; 89 Y27T; L253V; F254W; N348R; + 90 Y27T; D74P; L253V; F254W; A255G; N348R; + + 91 Y27T; D74G; L253V; F254W; A255G; W370I; ++ ++ 92 Y27T; L253V; F254W; A255G; + 93 Y27T; D74G; L253V; F254W; T384P; ++ + 94 Y27T; D74N; F254W; N348R; W370I; + + 95 Y27T; D74S; L253V; N348R; W370I; D381K; T384P; ++ + 96 Y27T; L253V; A255G; W370I; + + 97 Y27T; D74G; F254W; A255G; D381F; + + 98 Y27T; L253V; K369C; W370I; + 99 Y27T; L253V; F254W; N348R; D381F; + + 100 Y27T; L253V; F254W; A255G; N348R; W370I; + + 101 Y27T; D74S; L253V; A255G; T384P; + + 102 Y27T; D74N; L253V; N348R; W370I; D381W; T384P; ++ + 103 Y27T; D74P; L253V; N348R; + + 104 Y27T; D74G; F254W; A255G; N348R; K369C; + + W370I; D381F; 105 Y27T; D74P; F254W; A255G; N348R; K369C; W3701; + + 106 65/66 Y27T; D74N; L253V; F254W; W370I; D381K; ++ ++ 107 Y27T; D74G; L253V; T384P; + + 108 Y27T; D74S; N348R; W370I; + + 109 Y27T; D74S; L253V; F254W; A255G; K369C; W3701; + + 110 Y27T; D74G; L253V; N348R; K369C; W370I; + 111 Y27T; D74N; L253V; F254W; A255G; ++ + 112 Y27T; D74P; L253V; F254W; N348R; + + 113 Y27T; D74G; A255G; W370I; + + 114 Y27T; D74G; L253V; A255G; N348R; D381W; + + 115 Y27T; D74P; L253V; W370I; ++ + 116 Y27T; L253V; N348R; W370I; T384P; + 117 Y27T; D74G; L253V; F254W; A255G; W370I; D381K; T384P; ++ + 118 Y27T; D74N; F254W; A255G; N348R; W370I; D381K; + + 119 Y27T; D74S; L253V; N348R; K369C; W370I; T384P; + + 120 Y27T; D74N; L253V; F254W; N348R; W370I; D381F; ++ ++ 121 Y27T; D74S; F254W; A255G; N348R; W370I; + + D381F; T384P; 122 Y27T; D74G; L253V; F254W; A255G; D381K; ++ ++ 123 Y27T; D74S; F254W; K369L; W370I; + + 124 Y27T; L253V; F254W; D381F; T384P; + + 125 Y27T; D74G; L253V; F254W; A255G; K369C; + W3701; 126 Y27T; D74G; F254W; A255G; N348R; W370I; ++ + 127 Y27T; D74G; L253V; F254W; A255G; N348R; ++ ++ W370I; T384P; 128 L253V; N348R; W370I; + 129 Y27T; D74N; L253V; F254W; K369C; + + 130 Y27T; D74G; F254W; A255G; N348R; + 131 Y27T; D74S; L253V; N348R; D381W; + + 132 Y27T; D74S; L253V; N348R; + + 133 Y27T; L253V; F254W; A255G; N348R; + 134 Y27T; D74S; L253V; A255G; N348R; W370I; D3 81K; ++ ++ 135 Y27T; F254W; A255G; N348R; K369C; W370I; + + 136 Y27T; D74N; L253V; F254W; A255G; N348R; ++ +++ W370I; T384P; 137 Y27T; D74S; F254W; A255G; W370I; ++ ++ 138 Y27T; F254W; A255G; K369C; W370I; D381F; T384P; + 139 Y27T; L253V; F254W; D381F; ++ + 140 Y27T; D74N; F254W; N348R; + 141 67/68 Y27T; F254W; A255G; W370I; + 142 Y27T; D74N; A107V; A255G; N348R; K369C; W3701; + 143 Y27T; F254W; A255G; N348R; W370I; + 144 Y27T; D74N; F254W; + + 145 Y27T; D74G; L253V; D381F; T384P; + + 146 Y27T; D74P; L253V; A255G; + + 147 Y27T; D74S; L253V; F254W; A255G; N348R; + + 148 Y27T; D74S; F254W; A255G; N348R; K369C; W3701; + 149 Y27T; D74P; W370I; + 150 Y27T; L253V; F254W; T384P; + + 151 Y27T; D74N; L253V; F254W; A255G; W370I; +++ +++ 152 Y27T; D74G; L253V; N348R; W370I; ++ + 153 Y27T; F254W; A255G; N348R; W370I; T384P; + 154 Y27T; D74G; L253V; F254W; N348R; + + 155 Y27T; L253V; A255G; W370I; D381F; T384P; + 156 Y27T; L253V; A255G; N348R; D381K; + + 157 Y27T; L253V; F254W; N348R; W370I; T384P; + + 158 Y27T; D74N; F254W; N348R; W370I; D381K; + + 159 Y27T; D74N; L253V; A255G; N348R; K369C; W3701; + + 160 Y27T; L253V; F254W; N348R; D381W; T384P; + + 161 Y27T; L253V; F254W; A255G; G260C; N348R; + + D381F; T384P; 162 Y27T; D74P; L253V; F254W; N348R; D381F; T384P; + + 163 Y27T; D74G; L253V; N348R; K369C; W370I; D381F; + + 164 Y27T; D74N; L253V; N348R; + + 165 Y27T; D74G; L253V; A255G; N348R; T384P; + + 166 Y27T; D74N; L253V; A255G; W370I; ++ ++ 167 Y27T; D74N; A255G; N348R; W370I; + + 168 Y27T; L253V; N348R; D381F; T384P; + 169 Y27T; L253V; D381F; T384P; + 170 Y27T; D74P; L253V; F254W; N348R; W370I; D381W; T384P; ++ + 171 Y27T; L253V; F254W; A255G; N348R; W370I; T384P; + 172 Y27T; L253V; F254W; + + 173 Y27T; L253V; F254W; N348R; W370I; D381F; ++ ++ 174 Y27T; D74G; L253V; A255G; + + 175 Y27T; D74N; L253V; F254W; N348R; W370I; +++ +++ 176 Y27T; D74N; L253V; F254W; N348R; K369C; W370I; D381K; + + 177 Y27T; D74G; L253V; F254W; A255G; N348R; + + K369C; W370I; D381F; 178 Y27T; D74G; L253V; F254W; N348R; K369C; W3701; + + 179 Y27T; D74N; L253V; + + 180 Y27T; D74G; L253V; F254W; A255G; N348R; + + 181 Y27T; F254W; A255G; T384P; + 182 Y27T; F254W; A255G; ++ + 183 Y27T; L253V; F254W; A255G; N348R; D381F; T384P; + + 184 Y27T; F254W; A255G; N348R; + + 185 Y27T; D74N; F254W; T384P; + 186 Y27T; L253V; N348R; + + 187 Y27T; D74G; A255G; N348R; + 12 Y27T; F254W; A470V; + 13 Y27T; L253V; A255G; N348R; + 188 Y27T; D74N; L253V; K369C; D381K; T384P; + + 189 Y27T; D74G; L253V; A255G; N348R; D381F; ++ + 6 Y27T; A255G; W370I; + 190 Y27T; D74S; F254W; A255G; N348R; + 191  9/10 Y27T; D74S; L253V; F254W; N348R; D381W; T384P; +++ ++ 192 Y27T; D74N; F254W; A255G; N348R; + 193 Y27T; D74P; L253V; F254W; A255G; ++ + 194 Y27T; L253V; N348R; W370I; D381F; T384P; ++ ++ 195 Y27T; D74S; A255G; W370I; + 196 N388E; + ++ 197 A255P; + ¹Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 12 and defined as follows: “+” > than 2.0-fold but less than 5.0-fold increase; “++” > than 5.0-fold but less than 10.0-fold; “+++” > than 10.0-fold. ²Levels of increased selectivity were determined relative to the reference polypeptide of SEQ ID NO: 12 and defined as follows: “+” > than 2.0-fold but less than 5.0-fold increase; “++” > than 5.0-fold but less than 7.0-fold; “+++” > than 7.0-fold.

Example 7 Improvement in the Acylation of Insulin at the B29 Position Compared to SEQ ID NO: 12

Acylation of B29 by the four variants listed in Table 7.1 was tested at shake flask scale. The shake flask powders were produced as described in Example 4. The reactions were carried out in 96 well deep-well plates, each containing 200 μL of 0.2 M TRIS, pH 9.25, 20% acetonitrile, 10 g/L insulin, 17 g/L methyl phenylacetate, and 0.9 g/L lyophilized enzyme powder reconstituted in 10 mM TRIS, pH 7.5. The HTP plates were heat-sealed and incubated in Thermotron® shakers (3 mm throw, model # AJ185, Infors) at 30° C., 100 rpm, for 5 hours. The reactions were quenched with 200 μl acetonitrile and mixed for 5 minutes using a bench top shaker. The plates were then centrifuged at 4000 rpm for 5 minutes and loaded into an HPLC for analysis.

The percent conversion relative to SEQ ID NO:12 (Percent Conversion FOP) was calculated as the percent conversion of the product formed by the variant over the percent conversion produced by SEQ ID NO: 12. The results are shown in Table 7.1. The percent conversion was quantified by dividing the area of the product peak by the sum of the areas of the substrate, product and impurities/side product peaks as observed by the HPLC analysis.

The percent selectivity relative to SEQ ID NO:12 (Percent Selectivity FOP) was calculated as the percent selectivity of the product formed by the variant over the percent selectivity produced by SEQ ID NO: 12. The percent selectivity was calculated by dividing the area of the product peak by the sum of the areas of the product and impurities/side product peaks as observed by the HPLC analysis.

TABLE 7.1 Activity and Selectivity of Variants Acylating at the B29 Site Relative to SEQ ID NO: 12 Acylation Acylation Percent Percent Conversion Selectivity (FIOP)¹ at the (FIOP)² for the SEQ ID B29 Site B29 Site Variant NO: Amino Acid Differences (Relative to (Relative to SEQ NO: (nt/aa) (Relative to SEQ ID NO: 12) SEQ ID NO: 12) ID NO: 12) 638 101/102 D623R; + + 68 21/22 D709R; + +++ 639 105/106 D709K; + ++ 640 107/108 Y27T; V28A; G71H; D74G; K103E; W119Y; + +++ L253Y; F256R; N348H; T352K; A373R; S374T; S390K; G444N; A451K; N494D; Q547K; A616Y; S646D; ¹Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 12 and defined as follows: “+” > than 10.0-fold but less than 50.0-fold increase; “++” > than 50.0-fold but less than 100.0-fold; “+++” > than 100.0-fold. ²Levels of increased selectivity were determined relative to the reference polypeptide of SEQ ID NO: 12 and defined as follows: “+” > than 10.0-fold but less than 50.0-fold increase; “++” > than 50.0-fold but less than 100.0-fold; “+++” > than 100.0-fold.

Example 8 Improvements in the Acylation of Insulin at the B29 Position Compared to SEQ ID NO: 108 in High Throughput Screening

SEQ ID NO: 108 was selected as the next parent enzyme, based on the results described in Example 7. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the soluble lysate was generated as described in Example 3.

HTP reactions were carried out in 96 well deep-well plates. Each reaction well contained 200 μL of 0.1 M TRIS, pH 9.25, 20% acetonitrile, 25 g/L insulin, 17 g/L methyl phenylacetate and 10 μl HTP supernatant. The HTP plates were incubated in Thermotron® shakers (3 mm throw, model # AJ185, Infors) at 30° C., 100 rpm, for 3 hours. The reactions were quenched with 200 μl acetonitrile and mixed for 5 minutes using a bench top shaker. Then, 400 μl of water were added and mixed for 5 minutes using a bench top shaker. The plates were then centrifuged at 4000 rpm for 5 minutes and loaded into an HPLC for analysis.

The percent conversion relative to SEQ ID NO:108 (Percent Conversion FIOP) was calculated as the percent conversion of the product formed by the variant over the percent conversion produced by SEQ ID NO: 108. The results are shown in Table 8.1. The percent conversion was quantified by dividing the area of the product peak by the sum of the areas of the substrate, product and impurities/side product peaks as observed by the HPLC analysis.

TABLE 8.1 Activity of Variants Acylating at the B29 Site Relative to SEQ ID NO: 108 Acylation Percent SEQ ID Conversion (FIOP)¹ at Variant NO: Amino Acid Differences the A1 Site (Relative to NO: (nt/aa) (Relative to SEQ ID NO: 108) SEQ ID NO: 108) 301 25/26 R256V; ++++ 302 23/24 H71M; ++++ 303 27/28 K390A; ++++ 304 33/34 H71L; ++++ 305 31/32 R256L; +++ 306 35/36 R256K; +++ 307 29/30 K390R; +++ 308 K390L; +++ 309 K390P; ++ 310 K390C; ++ 311 H71Q; ++ 312 H71D; ++ 313 K390H; ++ 314 E707K; ++ 315 D623W; + 316 F50V; S619W; + 317 D252C; + 318 T705P; + 319 K369S; + 320 K369R; S619M; + 321 N185D; G415H; N444L; K723D; + 322 N185D; N444L; K723D; R748G; + 323 N9D; G415H; N444L; N457Q; K723D; R748E; + 324 N185D; K723D; R748E; + 325 K128H; K369R; + 326 N9D; T443D; N444K; K723D; R748G; A764E; + 327 S619F; + 328 N9D; N185D; G415H; K723D; R748E; + 329 S619M; + 330 N185D; R256H; T560G; R748E; + 331 N9D;K723D;R748S; + 332 K436G; + 333 N185D; N444K; N457Q; K668E; K723D; R748G; + 334 K128H; + 335 K723D; + 336 N185D; G415H; T443D; N444R; K723D; R748S; + 337 N444S; K723D; R748D; + 338 N185D; T560G; K723D; + 339 K128Q; + 340 N185D; R748D; + 341 N9D; N185D; + 342 T129W; N185D; N444L; K723D; R748E; + 343 N185D; N444K; R748S; + 344 N185D; G415H; N444S; R748E; + 345 A467V; + 346 F620L; + 347 N185D; T443D; N444S; K723D; R748E; + 348 N9D; G415H; T443D; N444L; K723D; R748E; + 349 Q626E; + 350 K723D; R748E; + 351 Q626M; + 352 N9D; T443D; N444S; K723D; R748E; + 353 D709E; + 354 K369W; + 355 G415H; N444S; T560G; K723D; R748S; + 356 G415H; K723D; + 357 T384E; + 358 N185D; N444K; K723D; + 359 N444L; K723D; + 360 K369R; T384Y; S619M; + 361 N9D; N185D; N444L; K723D; R748D; + 362 N457Q; K723D; R748D; + 363 K369R; H546P; + 364 N9D; N185D; G415H; N444L; R748E; + 365 K369Q; + 366 D623H; + 367 N9D; G415H; T443D; N444L; K723D; + 368 F254W; + 369 K369R; + 370 N185D; G415H; T443D; N444S; N457Q; R748E; + 371 N185D; G415H; N444S; K723D; R748S; + 372 N9D; N185D; N444S; K723D; R748E; + 373 N9D; N185D; T560G; K723D; R748E; + 374 K723D; R748D; + 375 T379N; + 376 N185D; G415H; N444L; T560G; K723D; + 377 N444K; K723D; R748E; + 378 N185D; T443D; N457Q; T560G; R748S; + 379 N185D; N457Q; R748S; + 380 N9D; N185D; K723D; R748S; + 381 N9D; G415H; T443D; N444L; R748S; + 382 N9D; N185D; G415H; T443D; + ¹Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 108 and defined as follows: “+” > than 1.2-fold but less than 2.0-fold increase; “++” > than 2.0-fold but less than 4.0-fold; “+++” > than 4.0-fold but less than 8.0-fold; “++++” > than 8.0-fold.

Example 9 Improvements in the Acylation of Insulin at the B29 Position Compared to SEQ ID NO: 24 in High Throughput Screening

SEQ ID NO: 24 was selected as the next parent enzyme, based on the results described in Example 8. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the soluble lysate was generated as described in Example 3.

HTP reactions were carried out in 96 well deep-well plates. Each reaction well contained 200 μL of 0.2 M TRIS, 20% acetonitrile, 25 g/L insulin, 17 g/L methyl phenylacetate and 10 μl HTP supernatant (the initial pH before the addition of lysate was 9.4). The HTP plates were incubated in Thermotron® shakers (3 mm throw, model # AJ185, Infors) at 30° C., 100 rpm, for 3 hours. The reactions were quenched with 200 μl acetonitrile and mixed for 5 minutes using a bench top shaker. Then, 400 μl of water were added and mixed for 5 minutes using a bench top shaker. The plates were then centrifuged at 4000 rpm for 5 minutes and loaded into an HPLC for analysis.

The percent conversion relative to SEQ ID NO: 24 (Percent Conversion FOP) was calculated as the percent conversion of the product formed by the variant over the percent conversion produced by SEQ ID NO: 24. The results are shown in Table 9.1. The percent conversion was quantified by dividing the area of the product peak by the sum of the areas of the substrate, product and impurities/side product peaks as observed by the HPLC analysis.

TABLE 9.1 Activity of Variants Acylating at the B29 Site Relative to SEQ ID NO: 24 Acylation Percent Conversion SEQ ID (FIOP)¹ at the A1 Variant NO: Amino Acid Differences Site (Relative to NO: (nt/aa) (Relative to SEQ ID NO: 24) SEQ ID NO: 24) 383 71/72 S67A; M71Q; N185D; R256L; T443D; Q626M; +++ 384 S67A; N185D; K390A; N444S; +++ 385 81/82 S67A; M71Q; R256L; T443D; S619W; +++ 386 S67A; N185D; K390A; T443D; Q626E; +++ 387 83/84 S67A; M71Q; N185D; R256L; S619W; +++ 388 75/76 S67A; M71Q; N185D; R256L; T443D; E707K; K723D; +++ 389 73/74 S67A; M71Q; R256L; K436G; T443D; S619W; Q626M; +++ 390 79/80 S67A; K390A; K436G; T443D; S619W; Q626E; +++ 391 77/78 M71Q; N185D; R256L; T443D; +++ 392 S67A; M71Q; N185D; R256L; +++ 393 S67A; M71F; K390A; S619W; ++ 394 S67A; K390A; N444S; S619F; ++ 395 S67A; K390A; ++ 396 M71Q; N185D; R256L; T443D; Q626E; K723D; ++ 397 S67A; M71F; N185D; K390A; S619W; ++ 398 N185D; K390A; T443D; ++ 399 S67A; M71Q; N185D; R256K; ++ 400 M71Q; R256L; T443D; ++ 401 M71Q; N185D; R256L; T443D; Q626M; E707K; ++ 402 S67A; M71F; K390A; T443D; S619F; Q626M; ++ 403 S67A; M71Q; N185D; R256L; Q626M; K723D; ++ 404 S67A; K390A; T443D; S619W; Q626E; ++ 405 M71Q; N185D; R256L; T443D; Q626E; E707K; ++ 406 M71L; V184Q; D252C; K369S; K390P; G415H; R748E; ++ 407 S67A; K390A; S619W; ++ 408 M71F; K390A; S619W; ++ 409 M71Q; R256L; K436G; E707K; ++ 410 S67A; K390A; K723D; ++ 411 S67A; M71Q; N185D; R256K; T384E; N444S; S619W; ++ 412 M71Q; N185D; R256K; S619F; ++ 413 S67A; M71Q; R256L; K436G; Q626M; ++ 414 K390A; ++ 415 N185D; K390A; K436G; Q626M; ++ 416 K390A; N444S; K723D; ++ 417 N185D; K390A; S619F; ++ 418 K369S; G415H; F620L; T705P; ++ 419 N185D; K390A; Q626M; ++ 420 M71Q; R256L; T443D; S619W; Q626E; ++ 421 M71Q; R256L; S619W; E707K; ++ 422 V184Q; D252C; K369S; K390P; R748E; ++ 423 M71Q; N185D; R256L; T384E; ++ 424 M71L; F254W; A638Q; D709E; R748S; ++ 425 S67A; M71L; D252C; K369S; K390P; ++ 426 K390A; T443D; S619W; ++ 427 M71Q; R256K; T443D; Q626M; ++ 428 K390A; N444S; S619W; ++ 429 S67A; M71Q; N185D; K390A; N444S; S619W; ++ 430 N185D; K390A; S619W; ++ 431 S67A; M71Q; N185D; K390A; S619W; E707K; ++ 432 N185D; K390A; Q626M; K723D; ++ 433 K128Q; K369S; T705P; R748E; ++ 434 M71Q; R256L; Q626M; E707K; ++ 435 K390A; K436G; S619W; K723D; ++ 436 K390A; K723D; ++ 437 K128Q; D252C; K369S; K390P; ++ 438 M71Q; N185D; R256L; T384E; S619W; ++ 439 M71Q; N185D; R256K; K390A; K436G; E707K; ++ 440 S67A; M71F; N185D; N444S; S619W; K723D; ++ 441 N185D; K436G; T443D; S619W; Q626M; E707K; K723D; ++ 442 M71Q; R256K; ++ 443 M71Q; R256L; K390L; T443D; ++ 444 S67A; M71F; K390L; T443D; Q626M; + 445 M71Q; R256L; K390L; T443D; Q626M; + 446 M71Q; N185D; K390A; T443D; S619W; Q626E; + 447 K128Q; K369S; G415H; + 448 K128H; F620L; T705P; R748E; + 449 M71L; T379N; D709E; R748G; + 450 T384E; P418G; N444S; A638Q; D709E; R748S; + 451 M71Q; N185D; K390A; N444S; + 452 M71F; N185D; K390L; N444S; S619W; K723D; + 453 M71L; D709E; + 454 N185D; R256K; S619W; E707K; + 455 K369S; K390P; F620L; + 456 M71Q; R256L; Q626E; E707K; + 457 S67A; M71F; N185D; N444S; S619W; + 458 K128Q; V184Q; D252C; K369S; G415H; + 459 F254W; T384E; A638Q; D709E; R748S; + 460 S67A; M71Q; Q134H; R256L; K390L; T443D; Q626M; E707K; + 461 K128Q; T705P; + 462 M71Q; N185D; R256L; K390L; + 463 S67A; T443D; S619R; + 464 M71Q; R256K; K390A; E707K; + 465 S67A; M71Q; T384E; K390A; N444S; S619W; + 466 M71L; F254W; N309T; N444S; + 467 M71L; P418G; R748S; + 468 M71Q; N185D; R256L; K390L; Q626M; + 469 M71L; + 470 S67A; M71Q; N185D; R256K; K390L; S619W; + 471 S67A; M71F; N185D; K390L; S619W; + 472 M71Q; N185D; K390A; K436G; T443D; Q626M; + 473 G415H; F620L; + 474 M71L; V184Q; K369S; G415H; T705P; + ¹Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 24 and defined as follows: “+” > than 1.2-fold but less than 2.0-fold increase; “++” > than 2.0-fold but less than 4.0-fold; “+++” > than 4.0-fold.

Example 10 Effect of the Addition of a Histidine Tag to SEQ ID NO: 82

Acylation of B29 of SEQ ID NO: 82 as described in Example 9, and SEQ ID NO: 110, which contains a six histidine tag at the c-terminus were compared at shake flask scale. The shake flask powders were produced as described in Example 4. The reactions were carried out in 96 well deep-well plates, each containing 200 μL comprised of 0.2 M TRIS, pH 9.25, 20% acetonitrile, 25 g/L insulin, 17 g/L methyl phenylacetate, and 0.3 to 10 g/L lyophilized enzyme powder reconstituted in 10 mM TRIS, pH 7.5. The HTP plates were heat-sealed and incubated in Thermotron® shakers (3 mm throw, model # AJ185, Infors) at 30° C., 100 rpm, for 3 hours. The reactions were quenched with 200 μl acetonitrile and mixed for 5 minutes using a bench top shaker. Then, 400 μl of water were added and mixed for 5 minutes using a bench top shaker. The plates were then centrifuged at 4000 rpm for 5 minutes and loaded into an HPLC for analysis. FIG. 2 provides a graph showing the results. As indicated, the addition of the histidine tag had minimal effect on the enzyme relative to the non-histidine tagged version.

Example 11 Improvements in the Acylation of Insulin at the B29 Position Compared to SEQ ID NO: 110 in High Throughput Screening

SEQ ID NO: 110 was selected as the parent enzyme after the histidine tag was shown to have minimal impact on the activity of SEQ ID NO: 82. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the soluble lysate was generated as described in Example 3, but with the use of 400 uL lysis buffer instead of 200 uL.

HTP reactions were carried out in 96 well deep-well plates. Each reaction well contained 200 μL of 0.2 M TRIS, 20% acetonitrile, 25 g/L insulin, 17 g/L methyl phenylacetate, and 10 μl HTP supernatant (the initial pH before the addition of lysate was 9.4). The HTP plates were incubated in Thermotron® shakers (3 mm throw, model # AJ185, Infors) at 30° C., 100 rpm, for 2 hours. The reactions were quenched with 200 μl acetonitrile and mixed for 5 minutes using a bench top shaker. Then, 400 μl of water were added and mixed for 5 minutes using a bench top shaker. The plates were then centrifuged at 4000 rpm for 5 minutes and loaded into an HPLC for analysis.

The percent conversion relative to SEQ ID NO: 110 (Percent Conversion FIOP) was calculated as the percent conversion of the product formed by the variant over the percent conversion produced by SEQ ID NO: 110. The results are shown in Table 9.1. The percent conversion was quantified by dividing the area of the product peak by the sum of the areas of the substrate, product and impurities/side product peaks as observed by the HPLC analysis.

TABLE 11.1 Activity of Variants Acylating at the B29 Site Relative to SEQ ID NO: 110 Acylation Percent SEQ ID Conversion Variant NO: Amino Acid Differences (FIOP)¹ at the A1 Site NO: (nt/aa) (Relative to SEQ ID NO: 110) (Relative to SEQ ID NO: 110) 475 E482Q; ++ 476 E482M; ++ 477 E482Y; ++ 478 37/38 E482I; ++ 479 E482T; ++ 480 G54A; + 481 39/40 S386G; + 482 D484T; + 483 E482L; + 484 T32V; + 485 N444S; D709E; R748S; + 486 G54S; + 487 G415H;D709E; + 488 Q556G; + 489 Y616D; + 490 K128Q; D252C; G415H; N444S; D709E; R748S; + 491 N185D;D709E; + 492 Y616N; + 493 Y616G; + 494 L557P; + 495 S704T; + 496 Y616A; + 497 47/48 L557S; + 498 43/44 P496A; + 499 P496T; + 500 45/46 L557Q; + 501 L557R; + 502 S639G; + 503 49/50 S704A; + 504 Q233A; + 505 D334P; + 506 Q233D; + 507 P496R; + 508 S639A; + 509 T131D; + 510 T131E; + 511 Q112D; + 512 S639E; + 513 L557V; + 514 S639D; + 515 P496N; + 516 S740A; + 517 L225T; + 518 T131N; + 519 L557M; + 520 D484L; + ¹Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 110 and defined as follows: “+” > than 1.0-fold but less than 2.0-fold increase; “++” > than 2.0-fold but less than 4.0-fold; “+++” > than 4.0-fold but less than 8.0-fold; “++++” > than 8.0-fold.

Example 12 Improvements in the Acylation of Insulin at the B29 Position Compared to SEQ ID NO: 40 in High Throughput Screening

SEQ ID NO: 40 was selected as the next parent enzyme, based on the results described in Example 11. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the soluble lysate was generated as described in Example 3, but with the use 400 uL lysis buffer, instead of 200 uL.

HTP reactions were carried out in 96 well deep-well plates. Each reaction well contained 200 μL of 0.2 M TRIS, 20% acetonitrile, 25 g/L insulin, 17 g/L methyl phenylacetate, and 80 μl HTP supernatant (the initial pH before the addition of lysate was 9.4). The HTP plates were incubated in Thermotron® shakers (3 mm throw, model # AJ185, Infors) at 30° C., 100 rpm, for 2 hours. The reactions were quenched with 200 μl acetonitrile and mixed for 5 minutes using a bench top shaker. Then, 400 μl of water were added and mixed for 5 minutes using a bench top shaker. The plates were then centrifuged at 4000 rpm for 5 minutes and loaded into an HPLC for analysis.

The percent conversion relative to SEQ ID NO: 40 (Percent Conversion (FIOP)) was calculated as the percent conversion of the product formed by the variant over the percent conversion produced by SEQ ID NO: 40. The results are shown in Table 12.1. The percent conversion was quantified by dividing the area of the product peak by the sum of the areas of the substrate, product and impurities/side product peaks as observed by the HPLC analysis.

The percent selectivity relative to SEQ ID NO:40 (Percent Selectivity FOP) was calculated as the percent selectivity of the product formed by the variant over the percent selectivity produced by SEQ ID NO: 40. The percent selectivity was calculated by dividing the area of the product peak by the sum of the areas of the product and impurities/side product peaks as observed by the HPLC analysis.

TABLE 12.1 Activity and Selectivity of Variants Acylating at the B29 Site Relative to SEQ ID NO: 40 Acylation Acylation Percent Percent Conversion Selectivity (FIOP)¹ at the (FIOP)² for the SEQ ID B29 Site B29 Site Variant NO: Amino Acid Differences (Relative to (Relative to SEQ NO: (nt/aa) (Relative to SEQ ID NO: 40) SEQ ID NO: 40) ID NO: 40) 524 53/54 W370K; + + 525 57/58 D623V; + + 526 51/52 A28V; Y52L; L55I; T131E; L175H; Q233R; + + G415H; N444G; P513S; 527 55/56 D623L; + + 528 59/60 P366G; + + 529 A28V; G74D; T374S; S704A; + + 530 A28V; L175H; P513S; W619S; + + 531 P366S; + + 532 L175H; G415H; K723D; + + 533 H472A; + + 534 H348N; N444G; S704A; + + 535 T374S; Y616A; S704A; + + 536 T27Y; A28V; G74D; N185D; T374S; N444G; + + S704A; 537 A28R; + + 538 N185D; T374S; + + 539 Y52L; L55I; Q112D; T374S; N444G; + 540 H348N; + 541 Q380F; + 542 S150A; + 543 A362R; + 544 T374S; G415H; K547Q; + ¹Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 40 and defined as follows: “+” > than 1.0-fold but less than 2.0-fold increase; “++” > than 2.0-fold but less than 4.0-fold; “+++” > than 4.0-fold but less than 8.0-fold; “++++” > than 8.0-fold. ²Levels of increased selectivity were determined relative to the reference polypeptide of SEQ ID NO: 40 and defined as follows: “+” > than 1.0-fold but less than 2.0-fold increase; “++” > than 2.0-fold but less than 4.0-fold; “+++” > than 4.0-fold but less than 8.0-fold; “++++” > than 8.0-fold.

Example 13 Improvements in the Acylation of Insulin at the B29 Position Compared to SEQ ID NO: 56 in High Throughput Screening

SEQ ID NO: 56 was selected as the next parent enzyme, based on the results described in Example 12. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the soluble lysate was generated as described in Example 3, but with the use of 400 uL lysis buffer, instead of 200 uL.

HTP reactions were carried out in 96 well deep-well plates. Each reaction well contained 200 μL of 0.2 M TRIS, 10% acetonitrile, 50 g/L insulin, 17 g/L methyl phenylacetate, and 10 μl HTP supernatant (the initial pH before the addition of lysate was 9.4). The HTP plates were incubated in Thermotron® shakers (3 mm throw, model # AJ185, Infors) at 30° C., 100 rpm, for 3 hours. The reactions were quenched with 200 μl acetonitrile and mixed for 5 minutes using a bench top shaker. Then, 400 μl of water were added and mixed for 5 minutes using a bench top shaker. The plates were then centrifuged at 4000 rpm for 5 minutes and diluted another 2-fold into water before being loaded into an HPLC for analysis.

The percent conversion relative to SEQ ID NO: 56 (Percent Conversion FIOP) was calculated as the percent conversion of the product formed by the variant over the percent conversion produced by SEQ ID NO: 56 and shown in the table below. The percent conversion was quantified by dividing the area of the product peak by the sum of the areas of the substrate, product and impurities/side product peaks as observed by the HPLC analysis.

The percent selectivity relative to SEQ ID NO:56 (Percent Selectivity HOP) was calculated as the percent selectivity of the product formed by the variant over the percent selectivity produced by SEQ ID NO: 56. The results are shown in Table 13.1. The percent selectivity was calculated by dividing the area of the product peak by the sum of the areas of the product and impurities/side product peaks as observed by the HPLC analysis.

TABLE 13.1 Activity and Selectivity of Variants Acylating at the B29 Site Relative to SEQ ID NO: 56 Acylation Acylation Percent Percent Conversion Selectivity (FIOP)¹ at the (FIOP)² for the SEQ ID B29 Site B29 Site Variant NO: Amino Acid Differences (Relative to SEQ (Relative to SEQ NO: (nt/aa) (Relative to SEQ ID NO: 56) ID NO: 56) ID NO: 56) 561 Q112D;W619S; + 562 87/88 P366S; D484T; W619S; I708V; + 563 P366Q; D484T; K547H; W619S; + 564 W619S; + 565 P366G; D484T; W619S; F620L; + 566 95/96 K390Q; + 567 K390A; + 568 K390S; + 569 W619S; S740A; + 570 E103K; Q112D; A361G; P366G; D484T; W619S; + 571 Q157S; K723D; + 572 E103K; W619S; + 573 A361G; P366Q; D484T; W619S; F620L; + 574 Q157S; K436G; L623V; K723D; + 575 P366G; W619S; S740A; + 576 D484T; K547H; W619S; F620L; S740A; + 577 D484T; I708V; + 578 P366Q; Q556H; W619S; S740A; + 579 P366S; D484T; W619S; F620L; I708V; + 580 E103K; Q112L; P366Q; D484T; W619S; I708V; S740A; + 581 D130E; Q157S; L623T; Q626M; + 582 T131E; S704A; K723D; + 583 D130E; R317A; L623T; K723D; + 584 E561P; + 585 W370K; D484T; W619S; + 586 K436G; L623T; + 587 P366S; D484T; S740A; + 588 T131D; L557V; S639T; K723D; + 589 K369D; + 590 R317A; L623T; Q626M; K723D; + 591 D130E; Q157S; K436G; K723D; + 592 Y616T; + 593 K128D; D130E; Q157S; K436G; L623T; Q626M; + K723D; 594 L557P; + 595 91/92 A255G; + 596 P366S; D484T; + 597 Q112L; P366G; D484T; K547H; W619S; I708V; + 598 T131D; T491S; L557V; D709E; K723D; + 599 Q71G; + 600 D484T; I708V; S740A; + 601 S675E; + 602 P366S; W370K; D484T; Q556H; W619S; + 603 D130E; L623T; + 604 A255D; + 605 T131D; L557V; + 606 L225T; L557V; S704A; D709E; K723D; + 607 E103K; W370K; D484T; W619S; I708V; + 608 D484T; + 609 Q112D; P366S; D484T; + 610 Q112L; D484T; I708V; 611 97/98 F57H; + 612 99/100 F57C; + 614 89/90 A67S; + ¹Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 56 and defined as follows: “+” > than 1.2-fold but less than 2.0-fold increase; “++” > than 2.0-fold but less than 4.0-fold; “+++” > than 4.0-fold but less than 8.0-fold; “++++” > than 8.0-fold. ²Levels of increased selectivity were determined relative to the reference polypeptide of SEQ ID NO: 56 and defined as follows: “+” > than 1.2-fold but less than 2.0-fold increase; “++” > than 2.0-fold but less than 4.0-fold; “+++” > than 4.0-fold but less than 8.0-fold; “++++” > than 8.0-fold.

Example 14 Improvements in the Acylation of Insulin at the A1 Position Compared to SEQ ID NO: 70 in High Throughput Screening

SEQ ID NO: 70 was selected as an additional parent enzyme, based on the results described in Example 7. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the soluble lysate was generated as described in Example 3, but using 400 μl lysis buffer, instead of 200 μl.

HTP reactions were carried out in 96 well deep-well plates containing 200 μL of 0.1 M Tris-HCl, pH 9.25, 20% acetonitrile, 20 g/L insulin, 17 g/L methyl phenylacetate, and 10 μl HTP lysate. The HTP plates were incubated in Thermotron® shakers (3 mm throw, model # AJ185, Infors) at 30° C., 100 rpm, for 5 hours. The reactions were quenched with 200 μl acetonitrile and mixed for 5 minutes using a bench top shaker. The plates were then centrifuged at 4000 rpm for 5 minutes, diluted 2-fold into water, and loaded into an HPLC for analysis.

The percent conversion relative to SEQ ID NO:70 (Percent Conversion FIOP) was calculated as the percent conversion of the product formed by the variant over the percent conversion produced by SEQ ID NO: 70. The results are shown in Table 14.1. The percent conversion was quantified by dividing the area of the product peak by the sum of the areas of the substrate, product and impurities/side product peaks as observed by the HPLC analysis.

TABLE 14.1 Activity of Variants Acylating at the A1 Site Relative to SEQ ID NO: 70 Acylation Percent Conversion SEQ ID (FIOP)¹ at the A1 Variant NO: Amino Acid Differences Site (Relative to NO: (nt/aa) (Relative to SEQ ID NO: 70) SEQ ID NO: 70) 642 121/122 R317S; Q380P; G415H; T443D; A517K; T560G; F701W; +++ 643 119/120 A255P; Q380P; G444K; N457T; A517K; T560G; +++ 644 123/124 A255P; R317S; Q380P; G415H; T443D; G444S; A517K; T560G; F701W; +++ 645 113/114 A255P; R317S; Q380P; G415H; T443D; G444S; F454W; N457T; +++ A517K; T560G; F701W; 646 115/116 L55I; A255P; R317S; Q380P; T443D; G444S; F454W; H472P; +++ A517K; T560G; F701W; 647 111/112 A255P; R317S; Q380P; G415H; G444S; N457T; A517K; T560G; F701W; +++ 648 117/118 A255P; R317S; Q380P; G415H; T443D; F454W; N457T; A517K; T560G; F701W; +++ 649 A255P; R317S; Q380P; T443D; G444S; F454W; A517K; T560G; F701W; ++ 650 A255P; R317S; Q380P; T443D; G444S; N457T; T560G; F701W; ++ 651 A255P; R317S; Q380P; G444K; F454W; A517K; T560G; ++ 652 L253S; ++ 653 A255P; R317S; G415H; T443D; G444S; F454W; N457T; A517K; F701W; ++ 654 A255P; R317S; Q380P; G415H; T443D; F454W; N457T; A517K; ++ F701W; 655 A255P; R317S; Q380P; G444K; A517K; T560G; F701W; ++ 656 R317S; Q380P; T443D; G444K; F454W; A517K; T560G; F701W; ++ 657 Q556G; ++ 658 A255P; R317S; A373K; G415H; T443D; F454W; N457T; A517K; T560G; F701W; ++ 659 N185E; ++ 660 W370L; ++ 661 A255P; R317S; Q380P; G444S; N457T; A517K; T560G; F701W; ++ 662 A255P; R317S; A373K; G415H; G444K; N457T; A517K; T560G; F701W; ++ 663 A255P; R317S; Q380P; G444K; F454W; N457T; A517K; T560G; F701W; ++ 664 N348D; + 665 N348H; + 666 A255P; R317S; A373K; G415H; G444S; F454W; N457T; A517K; T560G; F701W; + 667 A255P; A373K; G415H; G444S; N457T; A517K; T560G; F701W; + 668 L557P; + 669 A362V; + 670 A255P; R317S; Q380P; T443D; F454W; T560G; F701W; + 671 E707A; + 672 A255P; R317S; Q380P; G444S; A517K; T560G; F701W; + 673 A255P; R317S; Q380P; G444K; N457T; A517K; T560G; F701W; + 674 T560G; + 675 A255P; R317S; G415H; G444S; F454W; A517K; T560G; F701W; + 676 R317S; A373K; G415H; T443D; F454W; A517K; T560G; F701W; + 677 A255P; R317S; K369T; G415H; T443D; N457T; A517K; F701W; + 678 A255P; R317S; A373K; G415H; G444K; N457T; A517K; S530D; F701W; + 679 L557G; + 680 A255P; R317S; G415H; F454W; N457T; A517K; T560G; F701W; + 681 A255P; R317S; A373K; G415H; G444K; F454W; A517K; T560G; F701W; + 682 A255P; R317S; Q380P; G444Q; F454W; A517K; T560G; F701W; + 683 L253R; + 684 A255P; R317S; Q380P; G444S; F454W; A517K; S530D; T560G; F701W; + 685 R317S; A373K; G415H; G444K; F454W; A517K; T560G; F701W; + 686 A255P; + 687 L557S; + 688 A255P; R317S; Q380P; G444K; F454W; N457T; A517K; F701W; + 689 S386G; + 690 L557E; + 691 F254G; + 692 A451K; + 693 A255P; R317S; Q380P; G444S; F454W; A517K; T560G; F701W; + 694 A255P; R317S; Q380P; G444K; F454W; T560G; F701W; + 695 F756P; + 696 R317S; Q380P; T443D; N457T; A517K; F701W; + ¹Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 70 and defined as follows: “+” > than 1.2-fold but less than 1.5-fold increase; “++” > than 1.5-fold but less than 2.0-fold; “+++” > than 2.0-fold.

Example 15 Improvements in the Acylation of Insulin at the A1 Position Compared to SEQ ID NO: 116 in High Throughput Screening

SEQ ID NO: 116 was selected as the next parent enzyme, based on the results described in Example 14. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the soluble lysate was generated as described in Example 3, but using 400 μl lysis buffer instead of 200 μl.

HTP reactions were carried out in 96 well deep-well plates containing 200 μL of 0.25 M Tris-HCl, pH 9.25, 20% acetonitrile, 50 g/L insulin, 17 g/L methyl phenylacetate, and 10 μl HTP lysate. The HTP plates were incubated in Thermotron® shakers (3 mm throw, model # AJ185, Infors) at 30° C., 100 rpm, for 4 hours. The reactions were quenched with 200 μl acetonitrile and mixed for 5 minutes using a bench top shaker. The plates were then centrifuged at 4000 rpm for 5 minutes, diluted 24× into water, and loaded into an HPLC for analysis.

The percent conversion relative to SEQ ID NO:116 (Percent Conversion FIOP) was calculated as the percent conversion of the product formed by the variant over the percent conversion produced by SEQ ID NO: 116 The results are shown in Table 15.1. The percent conversion was quantified by dividing the area of the product peak by the sum of the areas of the substrate, product and impurities/side product peaks as observed by the HPLC analysis.

TABLE 15.1 Activity of Variants Acylating at the A1 Site Relative to SEQ ID NO: 116 Acylation Percent Conversion SEQ ID (FIOP)¹ at the A1 Variant NO: Amino Acid Differences Site (Relative to NO: (nt/aa) (Relative to SEQ ID NO: 116) SEQ ID NO: 116) 697 125/126 L253S; N348D; G415H; N457T; Q556G; L557P; ++++ 698 131/132 N185E; L253S; N348H; G415H; N457T; Q556G; L557G; K723E; ++++ 699 L253S; S317R; N348H; A362V; G415H; N457T; Q556G; L557G; K723E; ++++ 700 133/134 N185E; L253S; S317R; N348D; A362V; ++++ 701 N185E; L253S; S317R; N348H; A362V; Q556G; L557G; ++++ 702 L253S; N348D; W370L; N457T; Q556G; L557G; ++++ 703 N185D; L253S; S317R; N348H; A362V; G415H; N457T; ++++ 704 N185E; L253S; S317R; G415H; N457T; L557P; K723E; ++++ 705 129/130 N185D; L253S; N348H; A362V; ++++ 706 L253S; S317R; N348D; A362V; G415H; N457T; ++++ 707 L253S; N348D; A362V; L557G; K723E; ++++ 708 L253S; N348D; A362V; N457T; L557G; K723E; ++++ 709 L253S; A362V; N457T; L557G; ++++ 710 L253S; N348D; A362V; G415H; L557G; ++++ 711 L253S; S317R; A362V; G415H; L557G; ++++ 712 L253S; S317R; N348H; A362V; W370L; Q556G; L557P; ++++ 713 L253S; S317R; N348D; A362V; G415H; Q556G; L557G; K723E; ++++ 714 L253S; N348D; L557P; ++++ 715 L253S; N348D; A362V; W370L; N457T; Q556G; L557G; K723E; ++++ 716 N185D; L253S; N348H; G415H; L557G; ++++ 717 N185E; L253S; N348H; A362V; K723E; ++++ 718 N185D; L253Q; G415H; N457T; L557G; ++++ 719 L253S; S317R; N348D; W370L; N457T; K723E; ++++ 720 L253S; S317R; N348D; G415H; L557G; ++++ 721 N185D; L253S; S317R; W370L; G415H; Q556G; L557G; ++++ 722 N185E; L253S; S317R; N348H; A362V; L557G; K723E; ++++ 723 N185D; L253S; S317R; N348H; L557G; R602C; ++++ 724 N185E; L253S; N348D; A362V; N457T; Q556G; L557P; ++++ 725 N185D; L253S; S317R; A362V; ++++ 726 L253S; S317R; N348D; A362V; N457T; Q556G; L557G; K723E; ++++ 727 N185D; L253S; N348H; A362V; G415H; ++++ 728 N185E; L253S; N348H; A362V; W370L; G415H; Q556G; L557G; ++++ 729 L253S; S317R; N348D; A362V; Q556G; L557P; ++++ 730 L253S; S317R; A362V; N457T; Q556G; L557G; K723E; ++++ 731 N185E; L253S; S317R; N348D; A362V; G415H; L557G; K723E; ++++ 732 L253S; S317R; N348D; L557P; ++++ 733 N185E; L253S; S317R; N348H; A362V; G415H; N457T; K723E; ++++ 734 137/138 N185D; L253S; S317R; N348H; A362V; G415H; ++++ 735 N185D; L253S; A362V; L557G; ++++ 736 N185D; L253Q; S317R; N348D; A362V; G415H; N457T; K723E; +++ 737 N185E; L253S; S317R; A362V; W370L; L557G; K723E; +++ 738 N185E; L253S; G415H; L557G; +++ 739 L253Q; N348D; N457T; Q556G; L557P; +++ 740 N185E; L253S; N348H; A362V; N457T; L557G; +++ 741 N185D; L253S; S317R; A362V; G415H; N457T; +++ 742 N185D; L253Q; S317R; N348D; W370L; N457T; +++ 743 N185D; L253Q; S317R; A362V; G415H; K723E; +++ 744 N185D; L253S; S317R; G415H; L557G; +++ 745 L253S; N348D; A362V; Q556G; L557G; +++ 746 L253S; S317R; A362V; +++ 747 L253S; S317R; G415H; N457T; +++ 748 L253S; S317R; N348D; A362V; N457T; Q556G; +++ 749 N185E; L253S; N348H; A362V; L557G; +++ 750 L253S; N348H; A362V; K723E; +++ 751 L253Q; N348D; A362V; N457T; L557P; +++ 752 L253S; S317R; N348D; A362V; +++ 753 N185E; L253S; N457T; L557P; +++ 754 N185E; L253Q; N348D; Q556G; L557G; +++ 755 L253S; N348D; A362V; W370L; L557P; K723E; +++ 756 L253Q; S317R; N348D; L557G; +++ 757 L253Q; A362V; W370L; N457T; Q556G; L557G; +++ 758 L253S; N348D; L557G; K723E; +++ 759 L253S; N348D; L557G; +++ 760 N185E; L253S; L557G; K723E; +++ 761 N185D; L253Q; S317R; Q556G; L557G; +++ 762 L253Q; S317R; A362V; N457T; Q556G; L557P; +++ 763 L253S; A362V; L557G; +++ 764 N185D; L253S; N348D; A362V; G415H; Q556G; K723E; +++ 765 L253S; S317R; N348H; N457T; Q556G; K723E; +++ 766 L253S; S317R; A362V; L557G; K723E; +++ 767 N185D; L253S; N457T; L557G; +++ 768 127/128 L253S; N348H; G415H; +++ 769 N185E; S317R; N348H; A362V; N457T; L557G; +++ 770 N185E; L253Q; N348H; A362V; N457T; +++ 771 L253S; S317R; N348D; K723E; +++ 772 L253S; A362V; G415H; +++ 773 N348D; A362V; N457T; Q556G; L557G; K723E; +++ 774 N185E; L253S; S317R; N348H; A362V; K723E; +++ 775 N185E; L253S; A362V; Q556G; K723E; +++ 776 L253S; S317R; A362V; Q556G; L557G; +++ 777 L253Q; N348D; A362V; Q556G; L557G; +++ 778 N185E; S317R; N348D; A362V; Q556G; L557P; +++ 779 N185E; L253S; S317R; N457T; +++ 780 N185E; L253S; S317R; N348H; G415H; L557G; K723E; +++ 781 T105N; N185D; L253Q; S317R; N348D; L557G; +++ 782 N185D; S317R; N348D; A362V; N457T; +++ 783 L253S; G415H; L557G; +++ 784 N185E; N348D; A362V; G415H; K723E; +++ 785 N185D; L253Q; N348D; G415H; N457T; L557G; K723E; +++ 786 N185E; L253Q; W370L; G415H; Q556G; L557G; +++ 787 N185D; A362V; G415H; Q556G; L557G; K723E; +++ 788 N185D; L253Q; S317R; N348D; N457T; +++ 789 L253Q; N348D; A362V; L557G; +++ 790 N185D; L253S; N348D; N457T; Q556G; L557G; K723E; +++ 791 N185E; S317R; N348H; N457T; Q556G; L557G; +++ 792 L253S; N348D; A362V; G415H; Q556G; L557P; +++ 793 N348D; A362V; G415H; L557G; +++ 794 N185D; L253S; A362V; +++ 795 N185D; A362V; W370L; Q556G; L557G; W701F; K723E; +++ 796 L253S; G415H; N457T; L557G; +++ 797 N185E; S317R; G415H; N457T; L557G; +++ 798 L253Q; S317R; N348D; A362V; G415H; +++ 799 N185E; L253S; A362V; +++ 800 L253S; L557P; +++ 801 L253Q; S317R; N348H; A362V; G415H; L557G; +++ 802 N185E; L253Q; S317R; N348D; +++ 803 L253S; S317R; N348H; W370L; G415D; N457T; K723E; +++ 804 L253Q; S317R; N348D; A362V; N457T; Q556G; K723E; +++ 805 N185D; S317R; N348D; G415H; L557G; +++ 806 L253Q; S317R; N348D; N457T; Q556G; L557G; K723E; +++ 807 N185D; L253S; N348H; +++ 808 N185E; S317R; N348H; W701F; K723E; +++ 809 N185E; N348D; A362V; W370L; G415H; K723E; +++ 810 S317R; A362V; N457T; L557G; K723E; +++ 811 N185D; N348H; A362V; Q556G; L557G; K723E; +++ 812 L253S; S317R; A362V; K723E; +++ 813 N185E; N348D; N457T; Q556G; K723E; +++ 814 L253S; N348H; A362V; +++ 815 L253S; N348D; +++ 816 S317R; N348D; A362V; G415H; +++ 817 L253Q; N348D; A362V; L557G; K723E; +++ 818 L253Q; N348D; G415H; L557G; K723E; +++ 819 L253Q; N348D; A362V; G415H; L557P; +++ 820 135/136 L253S; S317R; N348D; +++ 821 N185D; L253Q; S317R; N348D; L557G; K723E; +++ 822 L253S; S317R; L557G; K723E; +++ 823 N185E; S317R; N348D; A362V; G415H; L557P; +++ 824 L253S; Q556G; L557G; K723E; +++ 825 N185E; L253S; S317R; +++ 826 N185E; L253Q; S317R; N348D; A362V; K723E; +++ 827 L253Q; S317R; N348D; A362V; Q556G; K723E; +++ 828 N185E; L253S; N457T; L557P; R602C; +++ 829 L253Q; A362V; Q556G; L557G; K723E; +++ 830 N185D; N348D; A362V; +++ 831 N185E; L253S; S317R; A362V; +++ 832 L253Q; S317R; A362V; L557G; +++ 833 N185D; N348D; L557G; +++ 834 N185D; L253Q; S317R; N457T; +++ 835 S317R; N348D; A362V; N457T; L557G; +++ 836 L253Q; S317R; G415H; L557P; K723E; +++ 837 N185D; N348D; W370L; G415H; Q556G; L557P; K723E; +++ 838 K304E; S317R; N348H; A362V; L557G; W701F; +++ 839 N185D; L253S; +++ 840 N185E; G415H; N457T; Q556G; K723E; +++ 841 G415H; Q556G; +++ 842 L253S; N457T; L557P; +++ 843 S317R; N348D; G415H; N457T; +++ 844 L253Q; G415H; L557G; +++ 845 L253Q; S317R; N348D; +++ 846 N185D; N348H; A362V; L557P; +++ 847 N185D; L253S; N348D; A362V; W370L; G415H; D428G; Q556G; K723E; +++ 848 N348D; Q556G; L557G; +++ 849 N185D; N348D; N457T; K723E; +++ 850 N185E; L253Q; N348D; L557G; +++ 851 N185D; A362V; G415H; L557P; +++ 852 N185E; S317R; A362V; N457T; L557G; K723E; +++ 853 L253Q; S317R; G415H; Q556G; L557G; K723E; +++ 854 L253Q; N348H; A362V; L557G; +++ 855 N185E; N348D; A362V; L557P; +++ 856 L253Q; A362V; W701F; K723E; +++ 857 S317R; N348D; A362V; Q556G; L557P; K723E; +++ 858 N185E; L253S; K723E; +++ 859 N185D; S317R; N348D; +++ 860 N185D; S317R; N348H; A362V; +++ 861 N185E; S317R; N457T; Q556G; L557G; +++ 862 N185E; N348D; A362V; +++ 863 S317R; A362V; L557G; +++ 864 N185E; A362V; N457T; +++ 865 N185D; L253Q; S317R; N348H; K723E; +++ 866 N348H; A362V; N457T; W701F; K723E; +++ 867 N348D; A362V; N457T; +++ 868 N185D; S317R; N348H; N457T; +++ 869 N185E; N457T; Q556G; +++ 870 N185E; S317R; N348D; G415H; L557P; W701F; +++ 871 L253S; N348H; N457T; +++ 872 N185E; L253Q; N348D; G415H; K723E; +++ 873 L253S; Q556G; L557G; +++ 874 N185E; L253Q; W701F; K723E; +++ 875 N185D; S317R; A362V; Q556G; +++ 876 L253S; N348H; K723E; +++ 877 N185E; L253Q; S317R; N457T; +++ 878 N185D; G415H; N457T; K723E; +++ 879 N185E; N348D; G415H; +++ 880 L253Q; N348H; A362V; W370L; L557P; +++ 881 N185D; G415H; Q556G; +++ 882 N185E; N348D; A362V; N457T; K723E; +++ 883 N185D; S317R; L557P; +++ 884 L253S; N348H; L557G; +++ 885 N185D; S317R; W370L; N457T; L557G; +++ 886 N185D; S317R; N348D; A362V; K723E; +++ 887 S317R; A362V; W370L; L557G; +++ 888 N185D; S317R; W701F; +++ 889 L253Q; G415H; N457T; +++ 890 L253S; A362V; +++ 891 N348H; G415H; Q556G; L557P; +++ 892 N185D; N348D; A362V; K723E; +++ 893 N185D; N348H; A362V; +++ 894 N185E; L253Q; S317R; A362V; +++ 895 N185E; L253Q; N348H; L557G; +++ 896 N185D; W701F; +++ 897 L253Q; S317R; A362V; G415H; +++ 898 L253Q; S317R; W370L; N457T; L557G; K723E; +++ 899 N185E; S317R; A362V; +++ 900 L253S; S317R; +++ 901 L253Q; N348H; L557G; +++ 902 N185D; L557G; K723E; +++ 903 N185E; G415H; L557G; +++ 904 N185E; W701F; +++ 905 S317R; N348D; L557G; +++ 906 L253S; N348H; N457T; Q556G; L557G; Y580N; K723E; +++ 907 N185E; A362V; N457T; K723E; +++ 908 N348D; A362V; L557G; K723E; +++ 909 L253Q; S317R; A362V; G415H; K723E; +++ 910 L253Q; N457T; Q556G; L557G; +++ 911 G415H; N457T; Q556G; L557G; +++ 912 L253Q; S317R; L557G; K723E; +++ 913 S317R; N457T; Q556G; L557G; +++ 914 N348H; G415H; N457T; +++ 915 N185D; L253Q; N348H; L557P; K723E; +++ 916 L253Q; A362V; N457T; K723E; +++ 917 N185D; L253Q; Q556G; L557P; K723E; +++ 918 N185D; G415H; K723E; +++ 919 S317R; A362V; N457T; +++ 920 L253Q; N348D; Q556G; K723E; +++ 921 S317R; L557G; K723E; +++ 922 N185E; Q556G; L557G; K723E; +++ 923 N185D; A362V; L557G; +++ 924 N185E; N348D; K723E; +++ 925 L253Q; A362V; N457T; +++ 926 L253S; Q556G; +++ 927 N185D; N348D; +++ 928 L253Q; S317R; N457T; +++ 929 N185E; L253Q; +++ 930 L253S; L557G; +++ 931 N185E; N348D; +++ 932 L253Q; N348D; A362V; K723E; +++ 933 N185D; A362V; G415H; K723E; +++ 934 N185E; A362V; Q556G; +++ 935 L253Q; L557G; K723E; +++ 936 S317R; N348H; L557G; +++ 937 S317R; A362V; G415H; +++ 938 N185E; L253Q; S317R; +++ 939 N185D; L253Q; L557G; +++ 940 G415H; L557G; +++ 941 S317R; N457T; L557G; +++ 942 A362V; L557G; K723E; +++ 943 L253Q; S317R; Q556G; +++ 944 N348D; G415H; +++ 945 N185D; L253Q; N348H; A362V; K723E; +++ 946 N348H; Q556G; +++ 947 A362V; N457T; L557G; +++ 948 L253Q; S317R; N348H; +++ 949 L253Q; N348D; W370L; Q556G; L557G; K723E; +++ 950 N185E; Q556G; L557P; +++ 951 A362V; W370L; L557G; K723E; +++ 952 L253Q; Q556G; L557G; +++ 953 L253S; N457T; +++ 954 N185E; S317R; N457T; K723E; +++ 955 S317R; A362V; Q556G; L557G; +++ 956 N185D; N348H; N457T; +++ 957 A362V; N457T; Q556G; L557P; +++ 958 G415H; L557P; +++ 959 L253S; +++ 960 N185E; L253Q; N457T; +++ 961 L253S; A642V; +++ 962 N348D; A362V; +++ 963 N185E; S317R; W370L; Q556G; +++ 964 L253Q; A362V; +++ 965 L253Q; N348H; A362V; +++ 966 N457T; Q556G; L557P; +++ 967 A160S; N185E; S317R; A362V; L557G; +++ 968 S317R; W370L; L557G; +++ 969 S317R; W370L; N457T; +++ 970 N457T; L557P; +++ 971 S317R; Q556G; +++ 972 A362V; N457T; +++ 973 S317R; A362V; +++ 974 S317R; Q556G; L557G; K723E; +++ 975 S317R; G415H; +++ 976 N185D; L253S; V360I; G415H; L557G; +++ 977 N348D; +++ 978 N348D; A362V; K723E; +++ 979 N185E; N348H; +++ 980 A362V; Q556G; L557P; +++ 981 N185D; N457T; +++ 982 N185D; A362V; +++ 983 L253Q; S317R; +++ 984 N348H; A362V; +++ 985 A362V; Q556G; K723E; +++ 986 N185E; A362V; +++ 987 A362V; L557G; +++ 988 S317R; Q556G; L557G; ++ 989 L253Q; K723E; ++ 990 N185D; S317R; ++ 991 S317R; N457T; ++ 992 S317R; L557G; ++ 993 G415H; ++ 994 A362V; N457T; K723E; ++ 995 N185D; K723E; ++ 996 Q556G; L557G; ++ 997 A362V; ++ 998 N185D; ++ 999 L253Q; S317R; ++ 1000 S317R; K723E; ++ 1001 Q556G; K723E; ++ 1002 L253Q; ++ 1003 L557G; ++ 1004 N185E; ++ 1005 S317R; ++ 1006 A140E; N185E; L253Q; G415H; N457T; L557G; ++ 1007 N457T; + 1008 L253S; S317R; A362V; N457T; K723E; S750R; + 1009 N185D; A362V; E707A; + 1010 L557P; + 1011 L253Q; F277L; L557P; + 1012 N185D; A362V; L557G; E707A; + 1013 N348H; + 1014 K723E; + 1015 N185D; S317R; A362V; E707A; + ¹Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 116 and defined as follows: “+” > than 1.2-fold but less than 1.5-fold increase; “++” > than 1.5-fold but less than 2.0-fold; “+++” > than 2.0-fold but less than 5.0-fold; “++++” > than 5.0-fold.

Example 16 Effect of the Addition of Histidine Tag to SEQ ID NO: 136

Acylation of A1 of SEQ ID NO: 136 (described in Example 15), and SEQ ID NO: 142 which contains a six histidine tag at the c-terminus of SEQ ID NO: 136 were compared at shake flask scale. The shake flask powders were produced as described in Example 4. Reactions were carried out in 96 well deep-well plates containing 200 μL of 0.25 M Tris-HCl, pH 9.25, 20% acetonitrile, 50 g/L insulin, 17 g/L methyl phenylacetate, and 0.05-0.5 g/L lyophilized enzyme powder. The plates were incubated in Thermotron® shakers (3 mm throw, model # AJ185, Infors) at 30° C., 100 rpm, for 4 hours. The reactions were quenched with 200 μl acetonitrile and mixed for 5 minutes using a bench top shaker. The plates were then centrifuged at 4000 rpm for 5 minutes, diluted 20× fold into water, and loaded into an HPLC for analysis. The results are shown in FIG. 3. As indicated by this Figure, the addition of the histidine tag had minimal effect on the enzyme relative to the non-histidine tagged version.

Example 17 Improvements in the Acylation of Insulin at the A1 and B29 Positions Compared to SEQ ID NO: 40 in High Throughput Screening

SEQ ID NO: 40 was selected as an additional parent enzyme, based on the results described in Example 11. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the soluble lysate was generated as described in Example 3.

HTP reactions were carried out in 96 well deep-well plates, each containing 200 μL comprised of 0.2 M TRIS, pH 9.25, 20% acetonitrile, 50 g/L insulin, 17 g/L methyl phenylacetate, and 10 μl HTP supernatant. The HTP plates were incubated in Thermotron® shakers (3 mm throw, model # AJ185, Infors) at 30° C., 100 rpm, for 2 hours. The reactions were quenched with 200 μl acetonitrile and mixed for 5 minutes using a bench top shaker. The plates were then centrifuged at 4000 rpm for 5 minutes and loaded into an HPLC for analysis.

The percent conversion relative to SEQ ID NO: 40 (Percent Conversion FIOP) was calculated as the percent conversion of the product formed by the variant over the percent conversion produced by SEQ ID NO: 40. The results are shown in Table 17.1. The percent conversion was quantified by dividing the area of the product peak by the sum of the areas of the substrate, product and impurities/side product peaks as observed by the HPLC analysis.

The percent selectivity relative to SEQ ID NO: 40 (Percent Selectivity FIOP) was calculated as the percent selectivity of the product formed by the variant over the percent selectivity produced by SEQ ID NO: 40. The results are shown in Table 17.1. The percent selectivity was calculated by dividing the area of the product peak by the sum of the areas of the product and impurities/side product peaks as observed by the HPLC analysis.

TABLE 17.1 Activity and Selectivity of Variants Acylating at the B29 Site Relative to SEQ ID NO: 40 Acylation Acylation Percent Percent Conversion Selectivity (FIOP)¹ at the (FIOP)² for the SEQ ID Al and B29 Al and B29 Sites Variant NO: Amino Acid Differences Sites (Relative to (Relative to SEQ NO: (nt/aa) (Relative to SEQ ID NO: 40) SEQ ID NO: 40) ID NO: 40) 1018 143/144 L55I; Q71G; A255P; N444S; T560G; ++++ +++ 1030 L55I; Q71G; K128W; Y253S; T560G; ++++ ++++ 1020 147/148 L55I; Q71G; G74D; N444S; ++++ +++ 1031 L256Y; N457M; G461R; ++++ ++++ 1032 L55I; Y253S; H348D; ++++ ++++ 1019 145/146 L55I; Q71G; K128W; N444S; ++++ +++ 1033 L55I; Q71G; G74D; ++++ +++ 1034 L55I; Q71G; ++++ +++ 1035 H348N; K352T; T384P; ++++ ++++ 1036 L55I; H348D; ++++ ++++ 1037 H348N; K352T; T384P; Q559H; ++++ ++++ 1038 L55I; Q71G; Y253S; A255P; ++++ +++ 1039 L55I; G74D; H348D; Q380P; N444S; T560G; ++++ +++ 1040 H348N; K352T; P364L; Y616R; ++++ ++++ 1041 L55I; Q71G; G74D; Y253S; A255P; ++++ +++ 1042 L55I; Q71G; G74D; A255P; H348D; ++++ +++ 1043 L55I; Q71G; G74D; A517K; ++++ +++ 1044 L55I; Q71G; G74D; N444S; F454W; A517K; ++++ ++++ 1023 153/154 L55I; Q71F; G74D; K390L; +++ ++ 1045 H348N; K352T; T384R; N457M; Y616R; +++ +++ 1046 L55I; Q71G; G74D; H348D; N444S; T560G; +++ +++ 1047 Q71M; G74S; N185D; K390A; Q626M; +++ ++ 1048 L55I; Y253S; Q380P; T560G; +++ ++ 1022 151/152 L55I; Q71M; G74S; Q380P; +++ ++ 1021 149/150 L55I; Q71M; Q380P; K436G; W619F; +++ ++ 1049 H348N; K352T; +++ +++ 1050 K128W; Y253S; F254W; A362V; N457T; +++ +++ Q556G; L557G; 1051 L55I; Q71F; W619F; +++ ++ 1052 L55I; Q71G; H348D; F701W; +++ +++ 1053 L55I; Q71M; G74D; N185D; +++ + 1054 L55I; Q71M; N185D; K436G; W619F; +++ + 1055 L55I; Y253S; N444S; +++ ++ 1056 H348N; K352T; T384R; +++ +++ 1057 L55I; Q71F; +++ ++ 552 L55I; H348D; Q380P; +++ + 1058 T129W; H348N; K352T; +++ +++ 1059 L55I; Q71M; Q380P; +++ + 1060 T129W; G202A; F254K; K352T; R373K; +++ +++ T384R; N457M; 1061 N185E; K369C; N457T; Q556G; L557G; +++ ++ D709E; K723E; 1062 L55I; N444S; +++ + 1063 N185E; G415H; N457T; K723E; +++ + 1064 A67S; Q71F; A255P; L256K; T384E; +++ ++++ 1065 Q71F; L256K; N444S; T560G; +++ ++ 1066 L256Y; H348N; T384R; +++ +++ 1067 Q71F; N444K; Y616E; +++ ++ 1068 H546L; + +++ 1069 N440L; + + 1070 D518R; + +++ 1071 A279P; + + 1072 A349E; + + 1073 N440Y; + + 1074 K682A; + + 1075 N333S; + + 1076 K682G; + + 1077 N333A; + + ¹Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 40 and defined as follows: “+” > than 1.2-fold but less than 5.0-fold increase; “++” > than 5.0-fold but less than 10.0-fold; “+++” > than 10.0-fold but less than 20.0-fold; “++++” > thand20.0-fold. ²Levels of increased selectivity were determined relative to the reference polypeptide of SEQ ID NO: 40 and defined as follows: “+” > than 1.2-fold but less than 5.0-fold increase; “++” > than 5.0-fold but less than 10.0-fold; “+++” > than 10.0-fold but less than 20.0-fold; “++++” > than 20.0-fold.

Example 18 Improvements in the Acylation of Insulin at the A1 and B29 Positions Compared to SEQ ID NO: 154 in High Throughput Screening

SEQ ID NO: 154 was selected as the next parent enzyme, based on the results described in Example 17. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the soluble lysate was generated as described in Example 3.

HTP reactions were carried out in 96 well deep-well plates, each containing 200 μL comprised of 0.2 M TRIS, pH 9.25, 20% acetonitrile, 50 g/L insulin, 17 g/L methyl phenylacetate, and 10 μl HTP supernatant. The HTP plates were incubated in Thermotron® shakers (3 mm throw, model # AJ185, Infors) at 30° C., 100 rpm, for 2 hours. The reactions were quenched with 200 μl acetonitrile and mixed for 5 minutes using a bench top shaker. The plates were then centrifuged at 4000 rpm for 5 minutes and loaded into an HPLC for analysis.

The percent conversion relative to SEQ ID NO: 154 (Percent Conversion FIOP) was calculated as the percent conversion of the product formed by the variant over the percent conversion produced by SEQ ID NO: 154. The results are shown in Table 18.1. The percent conversion was quantified by dividing the area of the product peak by the sum of the areas of the substrate, product and impurities/side product peaks as observed by the HPLC analysis.

The percent selectivity relative to SEQ ID NO: 154 (Percent Selectivity FIOP) was calculated as the percent selectivity of the product formed by the variant over the percent selectivity produced by SEQ ID NO: 154. The results are shown in Table 18.1. The percent selectivity was calculated by dividing the area of the product peak by the sum of the areas of the product and impurities/side product peaks as observed by the HPLC analysis.

TABLE 18.1 Activity and Selectivity of Variants Acylating at the B29 Site Relative to SEQ ID NO: 154 Acylation Acylation Percent Percent Conversion Selectivity (FIOP)¹ at the Al (FIOP)² for the SEQ ID and B29 Sites Al and B29 Sites Variant NO: Amino Acid Differences (Relative to SEQ (Relative to SEQ NO: (nt/aa) (Relative to SEQ ID NO: 154) ID NO: 154) ID NO: 154) 1026 159/160 N185E; H348D; K352T; L390A; N444S; +++ ++ 1027 163/164 F71G; F254W; H348D; T384P; L390A; N444S; +++ ++ 1078 Y253S; H348D; T384P; L390A; N444S; N457T; +++ ++ 1079 F71G; N185E; Y253S; F254W; H348D; K352T; T384P; L390A; N444S; +++ ++ 1028 161/162 N185E; Y253S; F254W; H348N; K352T; +++ ++ L390A; T560G; 1029 165/166 F71G; Y253S; H348D; K352T; T384P; L390A; T560G; +++ + 1080 H348D; K352T; L390A; N444S; +++ + 1081 F254W; H348N; T384P; L390A; +++ ++ 1082 K352T; L390A; N444S; ++ ++ 1083 H348D; K352T; L390A; ++ + 1084 N185E; F254W; H348N; K352T; T384P; ++ + L390A; N444S; 1085 Y253S; F254W; K352T; T384P; L390A; ++ + 1086 F254W; T384P; L390A; ++ + 1087 F71G; E165G; N185E; Y253S; F254W; H348D; ++ + K352T; L390A; N444S; 1088 F71G; K352T; L390A; ++ + 1089 F71G; T384P; L390A; N444S; T560G; ++ + 1090 F71G; N185E; F254W; H348D; K352T; L390A; N444S; ++ + 1091 F71G; F254W; H348N; K352T; T384P; L390A; N444S; ++ + 1092 G202A; F254W; H348D; T384P; L390A; N444S; ++ + 1093 Y253S; H348D; T384P; L390A; N444S; ++ + 1094 F71G; A466M; ++ + 1095 F71G; Y253S; K352T; T384P; L390A; T560G; ++ + 1096 H348D; L390A; N444S; ++ + 1097 F71G; H348N; T384P; L390A; T560G; ++ + 1098 F254W; K352T; L390A; ++ + 1099 Y253S; F254W; T384P; L390A; ++ + 1100 L390A; N444S; ++ + 1024 155/156 D74S; A362V; Q556G; ++ + 1101 F71G; Y253S; F254W; H348N; K352T; L390A; ++ + 1102 D74S; A362V; ++ + 1103 D74S; A362V; T384R; L557G; ++ + 1104 F71M; S251C; A279P; N333A; N444S; ++ + 1105 H348D; K352T; A362V; N444S; K723E; ++ + 1106 F71G; F254W; H348D; K352T; T384P; L390A; N444S; ++ + 1107 A279P; N440Y; N444S; ++ + 1108 F71G; A250S; A279P; N440L; N444S; A466M; A642E; ++ + 1109 F254W; H348N; L390A; ++ + 1110 D74S; A362V; N444K; ++ + 1025 157/158 D74G; N185E; K352T; Q380P; K436G; Y616R; ++ + 1111 D74S; ++ + 1112 D74S; A362V; N444S; Q556G; ++ + 1113 K352T; T384P; L390A; T560G; ++ + 1114 F71M; D74S; A362V; L390A; G415H; N444K; ++ + 1115 F71G; H348D; T384P; L390A; + + 1116 D74G; H348D; Q380P; K436G; K723E; + + 1117 F71G; P164S; Y253S; H348D; T384P; L390A; N444S; I637M; + + 1118 F71G; D74S; L390A; G415H; W619F; + + 1119 N185E; T384P; L390A; H472P; + + 1120 F254W; K352T; A362V; K723D; + + 1121 D74G; H348D; T560G; K723E; + + 1122 A67S; Y253S; F254W; H348D; K352T; G415H; K723D; + + 1123 F71G; H348D; K352T; T384P; L390A; N444S; + + 1124 F71G; H348D; L390A; + + 1125 F71G; N185E; H348N; L390A; + + 1126 N185D; H348D; K352T; K436G; T560G; K723D; + + 1127 F71G; F254W; T384P; L390A; N444S; T560G; + + 1128 D74S; A362V; T384P; + + 1129 A279P; N333A; A466M; + + 1130 F71G; Y253S; K352T; L390A; + + 1131 D74S; L557G; + + ¹Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 40 and defined as follows: “+” > than 1.2-fold but less than 2.0-fold increase; “++” > than 2.0-fold but less than 3.0-fold; “+++” > than 3.0-fold but less than 5.0-fold; “++++” > thand 5.0-fold. ²Levels of increased selectivity were determined relative to the reference polypeptide of SEQ ID NO: 40 and defined as follows: “+” > than 1.2-fold but less than 2.0-fold increase; “++” > than 2.0-fold but less than 3.0-fold; “+++” > than 3.0-fold but less than 5.0-fold; “++++” > thand 5.0-fold.

Example 19 Improvements in the Acylation of Insulin at the A1 and B1 Positions Compared to SEQ ID NO: 12 in High Throughput Screening

SEQ ID NO: 12 was selected as an additional parent enzyme, based on the results described in Example 7. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the soluble lysate was generated as described in Example 3.

HTP reactions were carried out in 96 well deep-well plates containing 200 μL of 0.1 M TRIS, pH 9.25, 20% acetonitrile, 10 g/L insulin, 17 g/L methyl phenylacetate, and 10 μl HTP supernatant. The HTP plates were incubated in Thermotron® shakers (3 mm throw, model # AJ185, Infors) at 30° C., 100 rpm, for 5 hours. The reactions were quenched with 200 μl acetonitrile or dimethylacetamide and mixed for 5 minutes using a bench top shaker. The plates were then centrifuged at 4000 rpm for 5 minutes and loaded into an HPLC for analysis.

The percent conversion relative to SEQ ID NO:12 (Percent Conversion FIOP) was calculated as the percent conversion of the product formed by the variant over the percent conversion produced by SEQ ID NO: 12. The results are shown in Table 19.1. The percent conversion was quantified by dividing the area of the product peak by the sum of the areas of the substrate, product and impurities/side product peaks as observed by the HPLC analysis.

TABLE 19.1 Activity and Selectivity of Variants Acylating at the B29 Site Relative to SEQ ID NO: 12 Acylation Acylation Percent Percent Conversion Selectivity (FIOP)¹ at the (FIOP)¹ for the SEQ ID A1 and B1 Sites A1 and B1 Sites Variant NO: Amino Acid Differences (Relative to (Relative to SEQ NO: (nt/aa) (Relative to SEQ ID NO: 12) SEQ ID NO: 12) ID NO: 12) 106 65/66 Y27T; D74N; L253V; F254W; W370I; D381K; ++++ +++ 141 67/68 Y27T; F254W; A255G; W370I; ++++ +++ 117 Y27T; D74G; L253V; F254W; A255G; W370I; D381K; T384P; ++++ +++ 120 Y27T; D74N; L253V; F254W; N348R; W370I; D381F; ++++ +++ 1132 Y27T; L253V; F254W; N348R; W370I; +++ ++ 157 Y27T; L253V; F254W; N348R; W370I; T384P; +++ ++ 1133 Y27T; D74N; L253V; F254W; A255G; N348R; +++ ++ W370I; D381W; T384P; 86 Y27T; D74N; L253V; F254W; N348R; W370I; +++ ++ D381K; T384P; 137 Y27T; D74S; F254W; A255G; W370I; +++ ++ 170 Y27T; D74P; L253V; F254W; N348R; W370I; D381W; T384P; +++ ++ 91 Y27T; D74G; L253V; F254W; A255G; W370I; +++ ++ 155 Y27T; L253V; A255G; W370I; D381F; T384P; +++ ++ 1134 Y27T; L253V; F254S; A255G; N348R; W370I; +++ ++ 258 Y27T; L253V; F254W; A255G; N348R; W370I; +++ ++ 1135 Y27T; L253V; F254W; A255G; N348R; W370I; +++ ++ 126 Y27T; D74G; F254W; A255G; N348R; W370I; +++ ++ 143 Y27T; F254W; A255G; N348R; W370I; +++ ++ 124 Y27T; L253V; F254W; D381F; T384P; +++ ++ 240 Y27T; L253V; F254W; +++ ++ 171 Y27T; L253V; F254L; A255G; N348R; W370I; T384P; +++ ++ 275 Y27T; D74G; L253V; F254W; +++ ++ 84 Y27T; D74G; L253V; F254W; +++ ++ 96 Y27T; L253V; A255G; W370I; ++ ++ 115 Y27T; D74P; L253V; W370I; ++ ++ 136 Y27T; D74N; L253V; F254W; A255G; N348R; W370I; T384P; ++ ++ 153 Y27T; F254W; A255G; N348R; W370I; T384P; ++ ++ 102 Y27T; D74N; L253V; N348R; W370I; D381R; T384P; ++ ++ 95 Y27T; D74S; L253V; N348R; W370I; D381K; ++ ++ 1136 Y27T; D74G; F254W; W370I; ++ ++ 122 Y27T; D74G; L253V; F254W; A255G; D381K; ++ ++ 145 Y27T; D74G; L253V; D381F; T384P; ++ ++ 116 Y27T; L253V; N348R; W370I; T384P; ++ ++ 154 Y27T; D74G; L253V; F254W; N348R; ++ ++ 1137 Y27T; L253V; N348R; W370I; T384P; ++ ++ 152 Y27T; D74G; L253V; N348R; W370I; ++ ++ 162 Y27T; D74P; L253V; F254W; N348R; D381F; T384P; ++ ++ 237 Y27T; D74G; L253V; N348R; W370I; ++ ++ 113 Y27T; D74G; A255G; W370I; ++ ++ 1138 Y27T; F254W; A255G; N348R; W370I; D381F; T384P; ++ + 208 Y27T; F254W; A255G; N348R; W370I; D381F; T384P; ++ + 643 119/120 K128W; A255P; Q380P; G444K; N457T; A517K; T560G; + + ¹Levels of increased activity or selectivity were determined relative to the reference polypeptide of SEQ ID NO: 12 and defined as follows: “+” > than 2.0-fold but less than 5-fold increase; “++” > than 5-fold but less than 10-fold; “+++” > than 10-fold but less than 15-fold: “++++” > than 15-fold.

Example 20 Improvements in the Acylation of Insulin at the B29 Position in the Presence of Phenylacetic Acid Compared to SEQ ID NO: 56 in High Throughput

SEQ ID NO: 56 was selected as an additional parent enzyme, based on the results described in Example 12. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the soluble lysate was generated as described in Example 3, but with the use of 400 uL lysis buffer instead of 200 uL.

HTP reactions were carried out in 96 well deep-well plates. Each reaction well contained 200 μL of 0.2 M TRIS, 10% acetonitrile, 50 g/L insulin, 17 g/L methyl phenylacetate, 12.5 or 15 g/L phenylacetic acid, and 10 μl HTP supernatant (the initial pH before the addition of lysate was 9.4). The HTP plates were incubated in Thermotron® shakers (3 mm throw, model # AJ185, Infors) at 30° C., 100 rpm, for 3 hours. The reactions were quenched with 200 μl acetonitrile and mixed for 5 minutes using a bench top shaker. Then, 400 μl of water were added and mixed for 5 minutes using a bench top shaker. The plates were then centrifuged at 4000 rpm for 5 minutes and diluted another 2×-fold into water before being loaded into an HPLC for analysis.

The percent conversion relative to SEQ ID NO: 56 (Percent Conversion FIOP) was calculated as the percent conversion of the product formed by the variant over the percent conversion produced by SEQ ID NO: 56. The results are shown in Table 20.1. The percent conversion was quantified by dividing the area of the product peak by the sum of the areas of the substrate, product and impurities/side product peaks as observed by the HPLC analysis.

TABLE 20.1 Activity and Selectivity of Variants Acylating at the B29 Site Relative to SEQ ID NO: 56 Acylation Percent Conversion (FIOP)¹ at Variant SEQ ID NO: Amino Acid Differences the B29 Site (Relative NO: (nt/aa) (Relative to SEQ ID NO: 56) to SEQ ID NO: 56) 615 T27S; ++ 611 97/98 F57H; + 614 89/90 A67S; + 616 A160C; + 612  99/100 F57C; + 617 F57V; + 618 S704A; + 619 R373I; + 620 T379S; + ¹Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 56 and defined as follows: “+” > than 1.2-fold but less than 2.0-fold increase; “++” > than 2.0-fold.

Example 21 Analytical Detection of Insulin and its Acylated Products

Data described in Examples 5-18 were collected using analytical methods in Tables 21.1, 21.2, 21.3, 21.4, and 21.5. The methods provided herein all find use in analyzing the variants produced using the present invention. However, it is not intended that the methods described herein are the only methods applicable to the analysis of the variants provided herein and/or produced using the methods provided herein. The results shown in FIG. 1 correspond to elution order of the compounds for these methods.

TABLE 21.1 Analytical Method Instrument Agilent HPLC 1200 series Column Ascends Express C18, 4.6 × 100 or 150 mm, 2.7 uM Gradient I (A: 0.05% TFA in water; B: 0.05% TFA in MeCN) Mobile Phase Time(min) % A 0.0 95 0.1 70 8, 8.5 or 9 50 8.1, 8.6 or 9.1 5 8.2, 8.7, or 9.2  95   9, 9.2 or 9.5 95 Gradient II ((A: 0.05% TFA in water;B: 0.05% TFA in MeCN) Time(min)c % A 0 70 7 or 8 50 7.1 or 8.1 70  9 or 10 70 Flow Rate 1.0 mL/min Run Time ~10 min Product Elution Insulin; A1-acylated insulin; B 29-acylated insulin; order B1-acylated insulin; di-A1, B29-acylated insulin; di-A1, B1-acylated insulin; di-B1, B29-acylated insulin; tri-A1, B1, B29-acylated insulin Column 40° C. Temperature Injection Volume 5 μL Detection UV 218 nm and 280 nm Detector: MWD (Agilent 1200 series); Slit = 4 nm; peak width > 0.1 min; Reference = 360; BW = 8

TABLE 21.2 Analytical Method Instrument Agilent HPLC 1290 series Column Waters Cortecs UPLC C18 2.1 × 50 mm, 1.6 uM Gradient (A: 0.05% TFA in water; B: 0.05% TFA in MeCN) for samples Mobile Phase Time(min) % A 0.0 72 2.5 50 2.51-2.7 10 2.71 72 3 72 Gradient (A: 0.05% TFA in water; B: 0.05% TFA in MeCN) for wash Time(min) % A 0.0 72 1.7 0 1.71-2 72 Flow Rate 0.9 mL/min Run Time 3 min Product Elution Insulin; A1-acylated insulin; B 29-acylated insulin; B1-acylated insulin; di- order A1, B29-acylated insulin; di-A1, B1-acylated insulin; di-B1, B29-acylated insulin; tri-A1, B1, B29-acylated insulin Column 40° C. Temperature Injection Volume 0.5 μL Detection UV 218 nm and 280 nm Detector: MWD (Agilent 1290 series); Slit = 4 nm; peak width > 0.1 min; Reference = 360; BW = 8

TABLE 21.3 Analytical Method Instrument Agilent HPLC 1200 series Column Ascends Express C18, 4.6 × 100 or 150 mm, 2.7 uM Gradient I (A: 0.05% TFA in water; B: 0.05% TFA in MeCN) Mobile Phase Time(min) % A 0.0 95 0.1 70 5, 6 or 8 50 5.5, 6.5, or 8.1 5 5.7 or 6.7 5 5.8, 6.8, or 8.2 95 6, 7, or 9 95 Flow Rate 1.0 mL/min Run Time ~10 min Product Elution Insulin; A1-acylated insulin; B 29-acylated insulin; B1-acylated insulin; di- order A1,B29-acylated insulin; di-A1, B1-acylated insulin; di-B1, B29-acylated insulin; tri-A1, B1, B29-acylated insulin Column 40° C. Temperature Injection Volume 5 μL Detection UV 218 nm and 280 nm Detector: MWD (Agilent 1200 series); Slit = 4 nm; peak width > 0.1 min; Reference = 360; BW = 8

TABLE 21.4 Analytical Method Instrument Agilent HPLC 1290 series Column Waters Cortecs UPLC C18 2.1 × 50 mm, 1.6 uM Gradient (A: 0.05% TFA in water; B: 0.05% TFA in MeCN) for samples Mobile Phase Time(min) % A 0.0 72 1.5 50 1.51-1.7 10 1.71 72 2 72 Flow Rate 0.9 mL/min Run Time 3 min Product Elution Insulin; A1-acylated insulin; B 29-acylated insulin; B1-acylated insulin; di- order A1, B29-acylated insulin; di-A1, B1-acylated insulin; di-B1, B29-acylated insulin; tri-A1, B1, B29-acylated insulin Column 40° C. Temperature Injection Volume 0.5 μL Detection UV 218 nm and 280 nm Detector: MWD (Agilent 1290 series); Slit = 4 nm; peak width > 0.1 min; Reference = 360; BW = 8

TABLE 21.5 Analytical Method Instrument 1290 Agilent UPLC system equipped with a quaternary pump and DAD UV detector or a Thermo Vanquish UPLC system equipped with a DAD UV detector. Column Waters Cortecs UPLC C18 column (50 × 2.1 mm, 1.6 μ) Gradient (A: 0.05% TFA in water; B: 0.05% TFA in MeCN) for samples Mobile Phase Time(min) % B 0.0 25 2.0 50 2.1 90 2.25 90 2.5 25 Flow Rate 1.0 mL/min Run Time 3.25 min Product Elution phenylacetic acid, 0.32 minutes; insulin, 0.78 minutes; A1-monoacyl-insulin, order 0.91 minutes; B29-monoacyl-insulin, 0.95 minutes; B1-monoacyl-insulin, 1.02 minutes; A1 ,B29-diacyl-insulin, 1.09 minutes; A1, B1-diacyl-insulin, 1.14 minutes; B1, B29-diacyl-insulin, 1.21 minutes; A1, B1, B29-triacyl-insulin, 1.38 minutes Column 40° C. Temperature Injection Volume 1.0 μL Detection UV detection (λ = 218 nm, Slit was 4 nm, peak width was > 0.1 min, reference was 360 nm)

Example 22 Improvements in the Acylation of Insulin at the A1 and B29 Positions Compared to SEQ ID NO: 160 in High Throughput Screening

SEQ ID NO: 160 was selected as an additional parent enzyme, based on the results described in Example 18. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the soluble lysate was generated as described in Example 3.

HTP reactions were carried out in 96well deep well plates, each containing 200 μL comprised of 0.5 M TRIS, pH 10.0, 20% acetonitrile, 50 g/L insulin, 17 g/L methyl phenylacetate and 5-10 μl HTP supernatant. The HTP plates were incubated in Thermotron® shakers (3 mm throw, model # AJ185, Infors) at 30° C., 100 rpm, for 2 hours. The reactions were quenched with 200l acetonitrile and mixed for 5 minutes using bench top shaker. The plates were then centrifuged at 4000 rpm for minutes and loaded into an HPLC for analysis.

The percent conversion relative to SEQ ID NO: 160 (Percent Conversion OP) was calculated as the percent conversion of the product formed by the variant over the percent conversion produced by SEQ ID NO: 160. The results are shown in Table 22.1. The percent conversion was quantified by dividing the area of the product peak by the sum of the areas of the substrate, product and impurities/side product peaks as observed by the HPLC analysis.

TABLE 22.1 Activity and Selectivity of Variants Acylating at the A1 and B29 Sites Relative to SEQ ID NO: 160 Acylation Percent Acylation Percent Conversion (FIOP)¹ at Selectivity (FIOP)² for SEQ ID Amino Acid Differences the Al and B29 Sites the Al and B29 Sites Variant NO: (Relative to SEQ ID NO: (Relative to SEQ ID (Relative to SEQ ID NO: (nt/aa) 160) NO: 160) NO: 160) 1139 S251G; + 1140 N44S; + + 1141 N44R; + 1142 N44M; + + 1143 F254H; + 1144 N44Q; + + 1145 S251A; + + 1146 R317A; + + 1147 D348H; + + 1148 F254V; + + 1149 F254L; + + 115 S444A; + + 1151 I55V; + 1152 S444K; + 1153 S444R; + + 1154 N44D; + + 1155 N44E; + + 1156 R317W; + + 1157 N44T; + + 1158 F254C; + + 1159 S444L; + + 116 N44H; + + 1161 N44I; + + 1162 A39T; + + 1163 F254T; + + 1164 F254G; + + 1165 167/168 K172R; D484T + + 1166 F254Y; + + 1167 S444T; + + 1168 F254S; ++ + 1169 169/17  S251T; ++ + 117 171/172 A466M; A47T; K723E; ++ + 1171 173/174 E185D; A279P; Q38P; A466M; ++ + 1172 175/176 F254W; +++ ++ 1173 177/178 F254A; +++ ++ 1174 179/18  A39S; +++ ++ ¹Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 160 and defined as follows: “+” > than 1.2-fold but less than 2.0-fold increase; “++” > than 2.0-fold but less than 3.0-fold; “+++” > than 3.0-fold but less than 5.0-fold; “++++” > than 5.0-fold. ²Levels of increased selectivity were determined relative to the reference polypeptide of SEQ ID NO: 160 and defined as follows: “+” > than 1.2-fold but less than 2.0-fold increase; “++” > than 2.0-fold but less than 3.0-fold; “+++” > than 3.0-fold but less than 5.0-fold; “++++” > than 5.0-fold.

Example 23 Improvements in the Acylation of Insulin at the B29 Position Compared to SEQ ID NO: 100 in High Throughput Screening

SEQ ID NO: 100 was selected as the next parent enzyme, based on the results described in Example 13. Libraries of engineered genes were produced using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the soluble lysate was generated as described in Example 3, but with the use of 400 uL lysis buffer, instead of 200 uL.

HTP reactions were carried out in 96well deep well plates. Each reaction well contained 200 μL of 0.5M TRIS, 20% acetonitrile, 50 g/L insulin, 17 g/L methyl phenylacetate and 40 HTP supernant (initial pH before the addition of lysate=10). The HTP plates were incubated in Thermotron® shakers (3 mm throw, model # AJ185, Infors) at 30° C., 100 rpm, for 5 hours. The reactions were quenched with 200 μl acetonitrile and mixed for 5 minutes using a bench top shaker. 400 μl of water is added and the plates are again mixed for 5 minutes using a bench top shaker. The plates were then centrifuged at 4000 rpm for 5 minutes and diluted another 2-fold into water before being loaded into an HPLC for analysis.

The percent conversion relative to SEQ ID NO: 100 (Percent Conversion FOP) was calculated as the percent conversion of the product formed by the variant over the percent conversion produced by SEQ ID NO: 100 and shown in the table below. The percent conversion was quantified by dividing the area of the product peak by the sum of the areas of the substrate, product and impurities/side product peaks as observed by the HPLC analysis.

TABLE 23.1 Activity and Selectivity of Variants Acylating at the B29 Site Relative to SEQ ID NO: 100 Acylation Percent Conversion (FIOP)¹ SEQ ID at the B29 Site Variant NO: Amino Acid Differences (Relative to SEQ ID NO: (nt/aa) (Relative to SEQ ID NO: 100) NO: 100) 1175 195/196 N444S; N457T; A466M; D709E; ++ 1176 201/202 N333A; A362V; N457T; Y616T; ++ 1177 193/194 A255G; K390Q; Y616T; S675E; ++ 1178 199/200 N333A; N444S; N457T; Y616T; D709E; ++ 1179 A255G; L557G; Y616T; ++ 1180 181/182 A255G; L557G; E561P; Y616T; S675E; ++ 1181 F254H; A255G; L557G; Y616T; ++ 1182 A255G; R373N; Y616T; ++ 1183 R373N; L557G; E561P; Y616T; S675E; ++ 1184 A160T; A255G; L557G; Y616T; ++ 1185 L557G; Y616T; S675E; ++ 1186 A160T; A255G; K390Q; L557G; S675E; ++ 1187 K390Q; Y616T; ++ 1188 C57H; A255G; K369D; K390Q; ++ 1189 N444S; N457T; D709E; ++ 1190 L557G; Y616T; + 1191 K390Q; L557G; + 1192 N333A; A362V; T384E; Y616T; + 1193 F254H; A255G; L557G; S675E; + 1194 C57H; A255G; L557G; S675E; + 1195 Q157S; R316H; A362V; K390A; N444S; T560G; + 1196 A28V; P249A; T560G; D709E; + 1197 K390Q; + 1198 C57H; A255G; L557G; E561P; S675E; + 1199 A160T; L557G; Y616T; + 1200 L256Y; N333A; N444S; N457T; T560G; + 1201 A255G; R373N; + 1202 Y616T; + 1203 R373N; E561P; + 1204 K369D; L557G; E561P; + 1205 L557G; S675E; + 1206 C57H; A255G; L557G; + 1207 A160T; F254H; K390Q; L557G; Y616T; + 1208 A255G; K369D; R373N; S675E; + 1209 V56L; A255G; Y616T; + 1210 N333A; N444S; T560G; + 1211 C57H; Y616T; + 1212 F254H; A255G; K369D; K390Q; + 1213 L557G; + 1214 K369D; E561P; S675E; + 1215 C57H; A255G; + 1216 N333A; N444S; T560G; D709E; + 1217 V56L; L557G; Y616T; + 1218 V56L; C57H; A255G; L557G; E561P; + 1219 L256Y; N333A; Y616T; D709E; + ¹Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 100 which showed 33.9 ± 1.4% conversion. Improvements in performance were defined as follows: “+” > than 1.2-fold but less than 2.0-fold increase; “++” > than 2.

TABLE 23.2 Activity and Selectivity of Variants Acylating at the B29 Site Relative to SEQ ID NO: 100 Acylation Percent Conversion (FIOP)¹ SEQ ID at the B29 Site Variant NO: Amino Acid Differences (Relative to SEQ ID NO: (nt/aa) (Relative to SEQ ID NO: 100) NO: 100) 1220 189/190 A28T; L557V; K723D; + 1221 187/188 C57H; A255G; L557G; Y616T; S675E; + 1222 197/198 A362V; N444S; N457T; + 1223 185/186 C57H; A255G; Y616T; S675E; + 1224 183/184 C57H; F254H; A255G; L557G; Y616T; + 1225 191/192 A28T; C57V; K723D; + 1226 E561P; Y616T; + 1227 A255G; S675E; + 1228 N333A; H348N; A362V; N444S; T560G; Y616T; + 1229 V56L; C57H; F254H; A255G; R373N; Y616T; S675E; + 1230 F254H; A255G; L557G; S675E; + 1231 Y616T; S675E; + 1232 V56L; C57H; E561P; Y616T; S675E; + 1233 N333A; A362V; N444S; D709E; + 1234 A28T; Q112D; + 1235 L557V; W619S; K723D; + 1236 L557G; Y616T; + 1237 C57H; F254H; A255G; S675E; + 1238 C57V; Q112D; Q157S; A160C; L557V; L623T; K723D; + 1239 C57H; L557G; Y616T; + 1240 A28T; C57V; L557V; + 1241 A362V; N444S; D709E; + 1242 Y616T; + 1243 L557V; W619S; L623T; + 1244 Q112D; A160C; W619S; + 1245 A255G; + 1246 P249A; A362V; N444S; A466M; D709E; + 1247 D484T; S704A; + 1248 F254H; K390Q; Y616T; + 1249 Q112D; Q157S; L557V; W619S; L623T; + 1250 D484T; L557V; L623T; + 1251 Q112D; Q157S; L623T; + 1252 C57H; A160T; A255G; Y616T; S675E; + 1253 L557V; L623T; K723D; + 1254 C57H; A255G; L557G; + 1255 L623T; + 1256 D484T; L623T; + 1257 Q112D; A160C; D484T; L557V; W619S; L623T; K723D; + 1258 A362V; + 1259 C57H; F254H; A255G; + 1260 C57H; R373N; D484T; L557G; S675E; + 1261 C57H; R373N; L557G; S675E; + 1262 C57H; A255G; R373N; + 1263 P249A; R316H; A362V; N444S; A466M; Y616T; + 1264 L557V; K723D; + ¹Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 100 which showed 52.1 ± 2.2% conversion. Improvements in performance were defined as follows: “+” > than 1.2-fold but less than 2.0-fold increase; “++” > than 2.

All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention(s). 

We claim:
 1. An engineered polynucleotide encoding an engineered penicillin G acylase capable of acylating insulin, wherein the polypeptide sequence of said penicillin G acylase is 90% or more identical to SEQ ID NO: 100, and wherein the residue at position X67 as compared to SEQ ID NO: 100 is an alanine.
 2. The engineered polynucleotide encoding the engineered penicillin G acylase of claim 1, wherein said penicillin G acylase comprises a sequence that is at least 90% or more identical to at least one sequence set forth in Table 5.1, 5.2, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 7.1, 8.1, 9.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 20.1, 22.1, 23.1, and/or 23.2.
 3. The engineered polynucleotide encoding the engineered penicillin G acylase of claim 1, wherein said penicillin G acylase comprises a sequence set forth in Table 5.1, 5.2, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 7.1, 8.1, 9.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 22.1, 23.1, and/or Table 23.2.
 4. The engineered polynucleotide encoding the engineered penicillin G acylase of claim 1, wherein said engineered penicillin G acylase comprises a polypeptide sequence selected from SEQ ID NOS:100 and
 110. 5. The engineered polynucleotide sequence of claim 1, wherein said sequence comprises a polynucleotide sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NOS: 1, 11, 23, 39, 55, 69, 81, 99, 107, 109, 115, 135, 141, 153, and/or
 159. 6. A vector comprising the polynucleotide sequence of claim
 1. 7. The vector of claim 6, further comprising at least one control sequence.
 8. A host cell comprising the vector of claim
 7. 9. A method for producing acylated insulin, comprising: providing the engineered penicillin G acylase of claim 1 and insulin; exposing said engineered penicillin G acylase and said insulin under conditions such that said engineered penicillin G acylase acrylates said insulin, thereby producing acylated insulin.
 10. The method of claim 9, wherein said acylation is conducted in the presence of methyl phenylacetate.
 11. The method of claim 9, wherein said acylation occurs at any of positions A1, B1, and/or B29 of said insulin.
 12. The method of claim 10, wherein said acylation occurs at any of positions A1, B1, and/or B29 of said insulin.
 13. The method of claim 9, wherein said acylation occurs at position A1 of said insulin.
 14. The method of claim 9, wherein said acylation occurs at position B1 of said insulin.
 15. The method of claim 9, wherein said acylation occurs at position B29 of said insulin.
 16. The method of claim 9, wherein said engineered penicillin G acylase produces more than 90% more acylated insulin as compared to the production of acylated insulin by the polypeptide of SEQ ID NO: 2, 4, 12, 24, 40, 56, 70, 82, 100, 108, 110, 116, 136, 142, 154 and/or
 160. 